Heterodimerized polypeptide

ABSTRACT

The present inventors produced a heterodimerized polypeptide having an Fc region formed from two polypeptides with different amino acid sequences (a first polypeptide and a second polypeptide), and succeeded in producing a heterodimerized polypeptide containing an Fc region with improved Fc region function compared to that of a homodimer in which the Fc region is composed of only the first polypeptide or only the second polypeptide by conventional technology.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of International ApplicationSerial No. PCT/JP2012/066665, filed on Jun. 29, 2012, which claims thebenefit of Japanese Patent Application Serial No. 2011-146778, filed onJun. 30, 2011, and Japanese Patent Application Serial No. 2011-288240,filed on Dec. 28, 2011.

TECHNICAL FIELD

The present invention provides antibody constant regions whose aminoacid sequence is modified from a naturally-occurring antibody constantregion, antibodies comprising such constant regions, pharmaceuticalcompositions comprising such antibodies, and methods for producing them.

BACKGROUND ART

Antibodies are drawing attention as pharmaceuticals since they arehighly stable in blood and have few side effects (Non-patent Documents 1and 2). Almost all antibody pharmaceuticals currently on the market areantibodies of the human IgG1 subclass. So far, many studies have beencarried out on antibody-dependent cellular cytotoxicity (hereinafter,referred to as ADCC) and complement-dependent cytotoxicity (hereinafter,referred to as CDC) which are effector functions of the IgG classantibodies; and in human IgG class, antibodies of the IgG1 subclass havebeen reported to have the highest ADCC activity and CDC activity(Non-Patent Document 3). Furthermore, antibody-dependent cell-mediatedphagocytosis (ADCP), which is phagocytosis of target cells mediated byIgG class antibodies, is also shown as one of the antibody effectorfunctions (Non-Patent Documents 4 and 5).

For an IgG antibody to exhibit ADCC, CDC, or ADCP, the antibody Fcregion must bind to an antibody receptor which is present on the surfaceof effector cells such as killer cells, natural killer cells, andactivated macrophages (hereinafter denoted as FcγR) and variouscomplement components. In humans, the FcγRIa, FcγRIIa, FcγRIIb,FcγRIIIa, and FcγRIIIb isoforms have been reported as the FcγR proteinfamily, and the respective allotypes have also been reported (Non-patentDocument 6).

Enhancement of cytotoxic effector functions such as ADCC, ADCP, and CDCis drawing attention as a promising means for enhancing the anti-tumoreffects of antibodies. The importance of FcγR-mediated effectorfunctions of antibodies for their antitumor effects has been reportedusing mouse models (Non-patent Documents 7 and 8). Furthermore,correlation was observed between clinical effects in humans and thehigh-affinity polymorphic allotype (V158) and the low-affinitypolymorphic allotype (F158) of FcγRIIIa (Non-patent Document 9). Thesereports showed that antibodies having an Fc region that has beenoptimized for specific FcγR-binding mediate a stronger effectorfunction, as a result demonstrate more effective antitumor effects.

The balance between the binding activities of an antibody towards anactivating receptor consisted of FcγRIa, FcγRIIa, FcγRIIIa, andFcγRIIIb, and an inhibitory receptor consisted of FcγRIIb is animportant element when optimizing antibody effector function. Use of anFc region that enhances binding activity to activating receptors anddecreases binding activity to inhibitory receptors may be able to conferantibodies with optimum effector functions (Non-patent Document 10).Conversely, use of an Fc region that has sustained or decreased bindingactivity to activating receptors and enhanced binding activity toinhibitory receptors may be able to confer immunosuppressive effect toantibodies (Non-patent Document 11). For the binding between an Fcregion and FcγR, several amino acid residues in the antibody hingeregion and CH2 domain, and the sugar chain added to Asn at position 297(EU numbering) which is bound to the CH2 domain have been shown to beimportant for the binding between the Fc region and FcγR (Non-patentDocuments 12, 13, and 14). There has been research on Fc region variantsthat have various FcγR-binding properties mainly at this binding site,and Fc region variants that have higher binding activities to activatingFcγR have been obtained (Patent Documents 1 and 2). For example, Lazaret al. have successfully increased human FcγRIIIa (V158) bindingapproximately 370-fold by substituting Ser at position 239, Ala atposition 330, and Ile at position 332 (EU numbering) of human IgG1 withAsp, Leu, and Glu, respectively (Non-patent Document 15 and PatentDocument 2). The ratio of FcγRIIIa-binding to FcγIIb-binding (A/I ratio)of this variant is increased approximately 9-fold as compared to that ofthe wild type. Furthermore, Lazar et al. have successfully enhanced thebinding to FcγIIb approximately 430-fold (Non-patent Document 16).Shinkawa et al. have successfully enhanced the FcγRIIIa-bindingapproximately 100-fold by removing fucose from the sugar chain added toAsn at position 297 (EU numbering) (Non-patent Document 17).

However, the functional modifications of antibody Fc regions reported sofar have limitations, and there is a demand for better functionalmodifications of Fc regions.

PRIOR ART DOCUMENTS Patent Documents

-   [Patent Document 1] WO 2000/042072-   [Patent Document 2] WO 2006/019447-   [Patent Document 3] WO 2009/041062-   [Patent Document 4] WO 2006/106905

Non-Patent Documents

-   [Non-patent Document 1] Nature Biotechnology, 23, 1073-1078 (2005)-   [Non-patent Document 2] Eur. J. Pharm. Biopharm, 59(3), 389-96    (2005)-   [Non-patent Document 3] Chemical Immunology, 65, 88 (1997)-   [Non-patent Document 4] Cancer Res., 68, 8049-8057 (2008)-   [Non-patent Document 5] Blood, 113, 3735-3743 (2009)-   [Non-patent Document 6] Immunol. Lett. 82, 57-65 (2002)-   [Non-patent Document 7] Pro. Nat. Acad. Sci. 95: 652-656 (1998)-   [Non-patent Document 8] Nature Medicine, 6: 443-446 (2000)-   [Non-patent Document 9] Blood 99:754-758 (2002)-   [Non-patent Document 10] Science, 310, 1510-1512 (2005)-   [Non-patent Document 11] Science, 291, 484-486 (2001)-   [Non-patent Document 12] Chemical Immunology, 65, 88 (1997)-   [Non-patent Document 13] Eur. J. Immunol. 23, 1098 (1993)-   [Non-patent Document 14] Immunology, 86, 319 (1995)-   [Non-patent Document 15] Pro. Nat. Acad. Sci., 103, 4005-4010 (2006)-   [Non-patent Document 16] Mol. Immun. 45, 3926-3933 (2008)-   [Non-patent Document 17] J. Biol. Chem., 278, 3466-3473 (2003)

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

The present invention was achieved in view of the above circumstances.An objective of the present invention is to provide polypeptides withimproved Fc region function over conventional homodimerized polypeptideshaving an Fc region; pharmaceutical compositions comprising thepolypeptides; therapeutic agents or preventive agents forimmunoinflammatory diseases comprising the pharmaceutical compositions;therapeutic agents or preventive agents for various types of cancers;and methods for producing them. Furthermore, an objective of the presentinvention is to provide methods of improving the Fc region function overconventional homodimerized polypeptides having an Fc region.

Means for Solving the Problems

The present inventors performed dedicated research to solve theabove-mentioned problems. As a result, the present inventors producedheterodimerized polypeptides having an Fc region consisting of twopolypeptides with different amino acid sequences (a first polypeptideand a second polypeptide), and thus successfully produced aheterodimerized polypeptide comprising an Fc region with improved Fcregion function over conventional homodimers in which the Fc region iscomposed of only the first polypeptide or only the second polypeptide.More specifically, the present invention provides [1] to [78] below:

[1] a polypeptide comprising an Fc region, wherein the polypeptide ischaracterized in that the Fc region is composed of a heterodimercomprising a first polypeptide and a second polypeptide, and wherein thepolypeptide is characterized in that a function of the Fc region isaltered compared to that of a polypeptide characterized in that the Fcregion is composed of a homodimer comprising only the first polypeptideand compared to that of a polypeptide characterized in that the Fcregion is composed of a homodimer comprising only the secondpolypeptide;[2] the polypeptide of [1], wherein at least one or more amino acidmutation is introduced into the Fc region;[3] the polypeptide of [1] or [2], wherein the amino acid mutationincludes at least one amino acid mutation that improves the Fc regionfunction when the mutation is introduced into only one of the Fc regionscompared to when the mutation is introduced into the Fc regions of boththe first polypeptide and the second polypeptide and when the mutationis not introduced;[4] the polypeptide of [2] or [3], wherein at least one or more aminoacid mutation is introduced into a CH2 domain of the Fc region;[5] the polypeptide of [4], wherein at least one or more amino acidmutations are introduced into an amino acid site selected from the groupconsisting ofAla at position 231 (EU numbering);Pro at position 232 (EU numbering);Glu at position 233 (EU numbering);Leu at position 234 (EU numbering);Leu at position 235 (EU numbering);Gly at position 236 (EU numbering);Gly at position 237 (EU numbering);Pro at position 238 (EU numbering);Ser at position 239 (EU numbering);Val at position 240 (EU numbering);Asp at position 265 (EU numbering);Val at position 266 (EU numbering);Ser at position 267 (EU numbering);His at position 268 (EU numbering);Glu at position 269 (EU numbering);Asp at position 270 (EU numbering);Pro at position 271 (EU numbering);Gln at position 295 (EU numbering);Tyr at position 296 (EU numbering);Ser at position 298 (EU numbering);Tyr at position 300 (EU numbering);Ser at position 324 (EU numbering);Asn at position 325 (EU numbering);Lys at position 326 (EU numbering);Ala at position 327 (EU numbering);Leu at position 328 (EU numbering);Pro at position 329 (EU numbering);Ala at position 330 (EU numbering);Pro at position 331 (EU numbering);Ile at position 332 (EU numbering);Glu at position 333 (EU numbering);Lys at position 334 (EU numbering);Thr at position 335 (EU numbering);Ile at position 336 (EU numbering); andSer at position 337 (EU numbering) in the CH2 domain of the Fc region;[6] the polypeptide of any one of [1] to [5], wherein the alteration ofFc region function is at least one or more alterations selected from thegroup consisting of enhancement of binding activity and improvement ofselectivity of binding to an Fcγ receptor;[7] the polypeptide of [6], wherein the alteration of Fc region functionis enhancement of binding activity to an Fcγ receptor;[8] the polypeptide of [7], wherein the Fcγ receptor is at least one ormore receptors selected from the group consisting of FcγRIa, FcγRIIa R,FcγRIIa H, FcγRIIb, and FcγRIIIa;[9] the polypeptide of [8], wherein the Fcγ receptor is FcγRIa;[10] the polypeptide of [9], wherein at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Region i of Tables 2-1 and 2-2 of this specification areintroduced into the amino acid sequences of the first polypeptide and/orthe second polypeptide constituting the Fc region;[11] the polypeptide of [9], wherein at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Region ii of Tables 2-1, 2-2, and 2-3 of this specificationare introduced into the amino acid sequences of the first polypeptideand/or the second polypeptide constituting the Fc region;[12] the polypeptide of [8], wherein the Fcγ receptor is FcγRIIa R;[13] the polypeptide of [12], wherein at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Region i of Tables 3-1 and 3-2 of this specification areintroduced into the amino acid sequences of the first polypeptide and/orthe second polypeptide constituting the Fc region;[14] the polypeptide of [12], wherein at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Region ii of Tables 3-1 and 3-2 of this specification areintroduced into the amino acid sequences of the first polypeptide and/orthe second polypeptide constituting the Fc region;[15] the polypeptide of [8], wherein the Fcγ receptor is FcγRIIa H;[16] the polypeptide of [15], wherein at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Region i of Table 4 of this specification are introducedinto the amino acid sequences of the first polypeptide and/or the secondpolypeptide constituting the Fc region;[17] the polypeptide of [15], wherein at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Region ii of Table 4 of this specification are introducedinto the amino acid sequences of the first polypeptide and/or the secondpolypeptide constituting the Fc region;[18] the polypeptide of [8], wherein the Fcγ receptor is FcγRIIb;[19] the polypeptide of [18], wherein at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Region i of Table 5 of this specification are introducedinto the amino acid sequences of the first polypeptide and/or the secondpolypeptide constituting the Fc region;[20] the polypeptide of [18], wherein at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Region ii of Table 5 of this specification are introducedinto the amino acid sequences of the first polypeptide and/or the secondpolypeptide constituting the Fc region;[21] the polypeptide of [8], wherein the Fcγ receptor is FcγRIIIa;[22] the polypeptide of [21], wherein at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Region i of Table 6 of this specification are introducedinto the amino acid sequences of the first polypeptide and/or the secondpolypeptide constituting the Fc region;[23] the polypeptide of [21], wherein at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Region ii of Table 6 of this specification are introducedinto the amino acid sequences of the first polypeptide and/or the secondpolypeptide constituting the Fc region;[24] the polypeptide of [6], wherein the alteration of Fc regionfunction is improvement of selectivity of binding activity to an Fcγreceptor;[25] the polypeptide of [24], wherein the improvement of selectivity ofbinding activity to an Fcγ receptor refers to selectivity between anactivating Fcγ receptor and an inhibitory Fcγ receptor;[26] the polypeptide of [25], wherein among the Fey receptors, theactivating Fcγ receptor is at least one or more receptors selected fromthe group consisting of FcγRIa, FcγRIIa R, FcγRIIa H, and FcγRIIIa, andthe inhibitory Fcγ receptor is FcγRIIb;[27] the polypeptide of [26], wherein the activating Fcγ receptor isFcγRIa, and the inhibitory Fcγ receptor is FcγRIIb, which ischaracterized in that the FcγRIa-binding activity is selectivelyenhanced compared to the FcγRIIb-binding activity;[28] the polypeptide of [27], wherein at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Region a of Tables 19-1, 19-2, 19-3, and 19-4 of thisspecification are introduced into the amino acid sequences of the firstpolypeptide and/or the second polypeptide constituting the Fc region;[29] the polypeptide of [27], wherein at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Region b of Tables 19-1, 19-2, 19-3, 19-4, and 19-5 of thisspecification are introduced into the amino acid sequences of the firstpolypeptide and/or the second polypeptide constituting the Fc region;[30] the polypeptide of [26], wherein the activating Fcγ receptor isFcγRIa, and the inhibitory Fcγ receptor is FcγRIIb, which ischaracterized in that the binding activity to FcγRIa is selectivelydecreased compared to the FcγRIIb-binding activity;[31] the polypeptide of [30], wherein at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Region c of Tables 23-1 and 23-2 of this specification areintroduced into the amino acid sequences of the first polypeptide and/orthe second polypeptide constituting the Fc region;[32] the polypeptide of [30], wherein at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Region d of Tables 23-1 and 23-2 of this specification areintroduced into the amino acid sequences of the first polypeptide and/orthe second polypeptide constituting the Fc region;[33] the polypeptide of [26], wherein the activating Fcγ receptor isFcγRIIa R, and the inhibitory Fcγ receptor is FcγRIIb, which ischaracterized in that the binding activity to FcγRIIa R is selectivelyenhanced compared to the FcγRIIb-binding activity;[34] the polypeptide of [33], wherein at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Region a of Table 20-1 of this specification are introducedinto the amino acid sequences of the first polypeptide and/or the secondpolypeptide constituting the Fc region;[35] the polypeptide of [33], wherein at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Region b of Tables 20-1, 20-2, and 20-3 of thisspecification are introduced into the amino acid sequences of the firstpolypeptide and/or the second polypeptide constituting the Fc region;[36] the polypeptide of [26], wherein the activating Fcγ receptor isFcγRIIa R, and the inhibitory Fcγ receptor is FcγRIIb, which ischaracterized in that the FcγRIIa R-binding activity is selectivelydecreased compared to the FcγRIIb-binding activity;[37] the polypeptide of [36], wherein at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Region c of Table 24-1 of this specification are introducedinto the amino acid sequences of the first polypeptide and/or the secondpolypeptide constituting the Fc region;[38] the polypeptide of [36], wherein at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Region d of Tables 24-1 and 24-2 of this specification areintroduced into the amino acid sequences of the first polypeptide and/orthe second polypeptide constituting the Fc region;[39] the polypeptide of [26], wherein the activating Fcγ receptor isFcγRIIa H, and the inhibitory Fcγ receptor is FcγRIIb, which ischaracterized in that the FcγRIIa H-binding activity is selectivelyenhanced compared to the FcγRIIb-binding activity;[40] the polypeptide of [39], wherein at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Region a of Table 21-1 of this specification are introducedinto the amino acid sequences of the first polypeptide and/or the secondpolypeptide constituting the Fc region;[41] the polypeptide of [39], wherein at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Region b of Tables 21-1, 21-2, and 21-3 of thisspecification are introduced into the amino acid sequences of the firstpolypeptide and/or the second polypeptide constituting the Fc region;[42] the polypeptide of [26], wherein the activating Fcγ receptor isFcγRIIa H, and the inhibitory Fcγ receptor is FcγRIIb, which ischaracterized in that the FcγRIIa H-binding activity is selectivelydecreased compared to the FcγRIIb-binding activity;[43] the polypeptide of [42], wherein at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Region c of Tables 25-1 and 25-2 of this specification areintroduced into the amino acid sequences of the first polypeptide and/orthe second polypeptide constituting the Fc region;[44] the polypeptide of [42], wherein at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Region d of Tables 25-1, 25-2, and 25-3 of thisspecification are introduced into the amino acid sequences of the firstpolypeptide and/or the second polypeptide constituting the Fc region;[45] the polypeptide of [26], wherein the activating Fcγ receptor isFcγRIIIa, and the inhibitory Fcγ receptor is FcγRIIb, which ischaracterized in that the FcγRIIIa-binding activity is selectivelyenhanced compared to the FcγRIIb-binding activity;[46] the polypeptide of [45], wherein at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Region a of Table 22-1 of this specification are introducedinto the amino acid sequences of the first polypeptide and/or the secondpolypeptide constituting the Fc region;[47] the polypeptide of [45], wherein at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Region b of Tables 22-1, 22-2, and 22-3 of thisspecification are introduced into the amino acid sequences of the firstpolypeptide and/or the second polypeptide constituting the Fc region;[48] the polypeptide of [26], wherein the activating Fcγ receptor isFcγRIIIa, and the inhibitory Fcγ receptor is FcγRIIb, which ischaracterized in that the FcγRIIIa-binding activity is selectivelydecreased compared to the FcγRIIb-binding activity;[49] the polypeptide of [48], wherein at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Region c of Tables 26-1 and 26-2 of this specification areintroduced into the amino acid sequences of the first polypeptide and/orthe second polypeptide constituting the Fc region;[50] the polypeptide of [48], wherein at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Region d of Tables 26-1, 26-2, 26-3, and 26-4 of thisspecification are introduced into the amino acid sequences of the firstpolypeptide and/or the second polypeptide constituting the Fc region;[51] the polypeptide of [1], [21], or [45], wherein at least one or moreamino acid mutations selected from the group consisting ofsubstitution of amino acid L at position 234 (EU numbering) with Y;substitution of amino acid L at position 235 (EU numbering) with Y or Q;substitution of amino acid G at position 236 (EU numbering) with W;substitution of amino acid S at position 239 (EU numbering) with M;substitution of amino acid H at position 268 (EU numbering) with D;substitution of amino acid D at position 270 (EU numbering) with E;substitution of amino acid S at position 298 (EU numbering) with A;substitution of amino acid K at position 326 (EU numbering) with D;substitution of amino acid A at position 327 (EU numbering) with D;substitution of amino acid L at position 328 (EU numbering) with W;substitution of amino acid A at position 330 (EU numbering) with M or K;andsubstitution of amino acid K at position 334 (EU numbering) with E or Lare introduced into the amino acid sequences of the first polypeptideand/or the second polypeptide constituting the Fc region;[52] the polypeptide of [1], [21], or [45], wherein a mutation isintroduced into at least one amino acid selected from Leu at position234, Leu at position 235, Gly at position 236, Ser at position 239, Hisat position 268, Asp at position 270, Ser at position 298, Lys atposition 326, Ala at position 327, Leu at position 328, Ala at position330, and Lys at position 334 (EU numbering) in the amino acid sequencesof the first polypeptide and/or the second polypeptide constituting theFc region;[53] the polypeptide of [1], [21], or [45], wherein a mutation isintroduced into at least one amino acid selected from Leu at position234, Leu at position 235, Gly at position 236, Ser at position 239, Hisat position 268, Asp at position 270, Ser at position 298, Ala atposition 327, Leu at position 328, and Lys at position 334 (EUnumbering) in the amino acid sequence of either polypeptide of the firstpolypeptide and the second polypeptide constituting the Fc region; and amutation is introduced into at least one amino acid selected from Asp atposition 270, Lys at position 326, Ala at position 330, and Lys atposition 334 (EU numbering) in the amino acid sequence of the otherpolypeptide;[54] the polypeptide of [1], [21], or [45], wherein at least one aminoacid mutation selected from the group consisting ofsubstitution of amino acid L at position 234 (EU numbering) with Y;substitution of amino acid L at position 235 (EU numbering) with Y or Q;substitution of amino acid G at position 236 (EU numbering) with W;substitution of amino acid S at position 239 (EU numbering) with M;substitution of amino acid H at position 268 (EU numbering) with D;substitution of amino acid D at position 270 (EU numbering) with E;substitution of amino acid S at position 298 (EU numbering) with A;substitution of amino acid A at position 327 (EU numbering) with D;substitution of amino acid L at position 328 (EU numbering) with W; andsubstitution of amino acid K at position 334 (EU numbering) with Lis introduced into the amino acid sequence of either polypeptide of thefirst polypeptide and the second polypeptide constituting the Fc region;and at least one amino acid mutation selected from the group consistingofsubstitution of amino acid D at position 270 (EU numbering) with E;substitution of amino acid K at position 326 (EU numbering) with D;substitution of amino acid A at position 330 (EU numbering) with M or K;andsubstitution of amino acid K at position 334 (EU numbering) with Eis introduced into the amino acid sequence of the other polypeptide;[55] the polypeptide of [1], [21], or [45], wherein any one set ofmutations of (i) to (vi) is introduced into the amino acid sequence ofeither polypeptide of the first polypeptide and the second polypeptideconstituting the Fc region, and any one set of mutations of (vii) to(ix) is introduced into the amino acid sequence of the otherpolypeptide:

-   -   (i) substitution of amino acid L at position 234 (EU numbering)        with Y;        -   substitution of amino acid L at position 235 (EU numbering)            with Y;        -   substitution of amino acid G at position 236 (EU numbering)            with W;        -   substitution of amino acid H at position 268 (EU numbering)            with D; and        -   substitution of amino acid S at position 298 (EU numbering)            with A;    -   (ii) substitution of amino acid L at position 234 (EU numbering)        with Y;        -   substitution of amino acid L at position 235 (EU numbering)            with Y;        -   substitution of amino acid G at position 236 (EU numbering)            with W;        -   substitution of amino acid H at position 268 (EU numbering)            with D;        -   substitution of amino acid D at position 270 (EU numbering)            with E; and        -   substitution of amino acid S at position 298 (EU numbering)            with A;    -   (iii) substitution of amino acid L at position 234 (EU        numbering) with Y;        -   substitution of amino acid L at position 235 (EU numbering)            with Q;        -   substitution of amino acid G at position 236 (EU numbering)            with W;        -   substitution of amino acid S at position 239 (EU numbering)            with M;        -   substitution of amino acid H at position 268 (EU numbering)            with D;        -   substitution of amino acid D at position 270 (EU numbering)            with E; and        -   substitution of amino acid S at position 298 (EU numbering)            with A;    -   (iv) substitution of amino acid L at position 234 (EU numbering)        with Y;        -   substitution of amino acid L at position 235 (EU numbering)            with Y;        -   substitution of amino acid G at position 236 (EU numbering)            with W;        -   substitution of amino acid H at position 268 (EU numbering)            with D;        -   substitution of amino acid S at position 298 (EU numbering)            with A; and        -   substitution of amino acid A at position 327 (EU numbering)            with D;    -   (v) substitution of amino acid L at position 234 (EU numbering)        with Y;        -   substitution of amino acid L at position 235 (EU numbering)            with Y;        -   substitution of amino acid G at position 236 (EU numbering)            with W;        -   substitution of amino acid S at position 239 (EU numbering)            with M;        -   substitution of amino acid H at position 268 (EU numbering)            with D;        -   substitution of amino acid S at position 298 (EU numbering)            with A; and        -   substitution of amino acid A at position 327 (EU numbering)            with D;    -   (vi) substitution of amino acid L at position 234 (EU numbering)        with Y;        -   substitution of amino acid L at position 235 (EU numbering)            with Y;        -   substitution of amino acid G at position 236 (EU numbering)            with W;        -   substitution of amino acid S at position 239 (EU numbering)            with M;        -   substitution of amino acid H at position 268 (EU numbering)            with D;        -   substitution of amino acid S at position 298 (EU numbering)            with A;        -   substitution of amino acid A at position 327 (EU numbering)            with D;        -   substitution of amino acid L at position 328 (EU numbering)            with W; and        -   substitution of amino acid K at position 334 (EU numbering)            with L;    -   (vii) substitution of amino acid K at position 326 (EU        numbering) with D;        -   substitution of amino acid A at position 330 (EU numbering)            with M; and        -   substitution of amino acid K at position 334 (EU numbering)            with E;    -   (viii) substitution of amino acid D at position 270 (EU        numbering) with E;        -   substitution of amino acid K at position 326 (EU numbering)            with D;        -   substitution of amino acid A at position 330 (EU numbering)            with M; and        -   substitution of amino acid K at position 334 (EU numbering)            with E;    -   (ix) substitution of amino acid D at position 270 (EU numbering)        with E;        -   substitution of amino acid K at position 326 (EU numbering)            with D;        -   substitution of amino acid A at position 330 (EU numbering)            with K; and        -   substitution of amino acid K at position 334 (EU numbering)            with E;            [56] a polypeptide comprising an Fc region, wherein the            polypeptide is characterized in that the Fc region is            composed of a heterodimer comprising a first polypeptide and            a second polypeptide, and wherein the polypeptide is            characterized in that a function of the Fc region is altered            compared to that of a polypeptide characterized in that the            Fc region is composed of a homodimer comprising only the            first polypeptide or compared to that of a polypeptide            characterized in that the Fc region is composed of a            homodimer comprising only the second polypeptide;            [57] the polypeptide of [56], wherein the alteration of Fc            region function is at least one or more alterations selected            from the group consisting of enhancement of binding            activity, reduction of binding, and improvement of binding            selectivity of the polypeptide to an Fcγ receptor;            [58] the polypeptide of [57], wherein the alteration of Fc            region function is additionally an alteration that improves            physicochemical stability;            [59] the polypeptide of [58], wherein the alteration that            improves physicochemical stability means that a polypeptide            characterized in that the Fc region is composed of a            heterodimer comprising a first polypeptide and a second            polypeptide has a higher Tm than that of a polypeptide            characterized in that the Fc region is composed of a            homodimer comprising only the first polypeptide or that of a            polypeptide characterized in that the Fc region is composed            of a homodimer comprising only the second polypeptide;            [60] the polypeptide of any one of [57] to [59], wherein the            alteration of Fc region function is enhancement of binding            activity to an Fcγ receptor, and the Fcγ receptor is at            least one or more receptors selected from the group            consisting of FcγRIa, FcγRIIa R, FcγRIIa H, FcγRIIb, and            FcγRIIIa;            [61] the polypeptide of [60], wherein the Fcγ receptor is            FcγRIa;            [62] the polypeptide of [61], wherein at least one or more            amino acid mutations selected from the group consisting of            the amino acid mutations described in Tables 31-1, 31-2, and            31-3 of this specification are introduced into the amino            acid sequences of the first polypeptide and/or the second            polypeptide constituting the Fc region;            [63] the polypeptide of [60], wherein the Fcγ receptor is            FcγRIIa R;            [64] the polypeptide of [63], wherein at least one or more            amino acid mutations selected from the group consisting of            the amino acid mutations described in Tables 32-1 and 32-2            of this specification are introduced into the amino acid            sequences of the first polypeptide and/or the second            polypeptide constituting the Fc region;            [65] the polypeptide of [60], wherein the Fcγ receptor is            FcγRIIa H;            [66] the polypeptide of [65], wherein at least one or more            amino acid mutations selected from the group consisting of            the amino acid mutations described in Tables 33-1 and 33-2            of this specification are introduced into the amino acid            sequences of the first polypeptide and/or the second            polypeptide constituting the Fc region;            [67] the polypeptide of [60], wherein the Fcγ receptor is            FcγRIIb;            [68] the polypeptide of [67], wherein at least one or more            amino acid mutations selected from the group consisting of            the amino acid mutations described in Tables 34-1 and 34-2            of this specification are introduced into the amino acid            sequences of the first polypeptide and/or the second            polypeptide constituting the Fc region;            [69] the polypeptide of [60], wherein the Fcγ receptor is            FcγRIIIa;            [70] the polypeptide of [69], wherein at least one or more            amino acid mutations selected from the group consisting of            the amino acid mutations described in Tables 35-1 and 35-2            of this specification are introduced into the amino acid            sequences of the first polypeptide and/or the second            polypeptide constituting the Fc region;            [71] the polypeptide of any one of [1] to [70], wherein an            amino acid alteration is additionally introduced to impart            difference in isoelectric points between the first            polypeptide and the second polypeptide;            [72] the polypeptide of [71], wherein the amino acid            alteration to confer difference in isoelectric points is            characterized by the introduction of at least one amino acid            mutation at an amino acid site selected from the group            consisting of Gly at position 137, Gly at position 138, Thr            at position 139, Lys at position 147, Ser at position 192,            Leu at position 193, Gln at position 196, Tyr at position            198, Ile at position 199, Asn at position 203, Lys at            position 214, Val at position 263, Glu at position 272, Lys            at position 274, Tyr at position 278, Lys at position 288,            Lys at position 290, Gly at position 316, Lys at position            317, Lys at position 320, Lys at position 324, Thr at            position 335, Ser at position 337, Lys at position 340, Leu            at position 358, Lys at position 360, Gln at position 362,            Ser at position 364, Ser at position 383, Asn at position            384, Gly at position 385, Gln at position 386, Pro at            position 387, Asn at position 390, Val at position 397, and            Val at position 422 (EU numbering) in the amino acid            sequence of the first polypeptide and/or the second            polypeptide;            [73] the polypeptide of [71], wherein the amino acid            alteration to confer difference in isoelectric points is            characterized by the introduction of a mutation to at least            one amino acid selected from the group consisting of Gln at            position 196, Ile at position 199, Val at position 263, Glu            at position 272, Gly at position 316, Leu at position 358,            Ser at position 364, Ser at position 383, Pro at position            387, and Val at position 397 (EU numbering) in the amino            acid sequence of either polypeptide of the first polypeptide            and the second polypeptide; and introduction of a mutation            to at least one amino acid selected from the group            consisting of Gly at position 137, Gly at position 138, Thr            at position 139, Lys at position 147, Ser at position 192,            Leu at position 193, Tyr at position 198, Ile at position            199, Asn at position 203, Lys at position 214, Lys at            position 274, Tyr at position 278, Lys at position 288, Lys            at position 290, Gly at position 316, Lys at position 317,            Lys at position 320, Lys at position 324, Thr at position            335, Ser at position 337, Lys at position 340, Leu at            position 358, Lys at position 360, Gln at position 362, Ser            at position 383, Asn at position 384, Gly at position 385,            Gln at position 386, Asn at position 390, and Val at            position 422 (EU numbering) in the amino acid sequence of            the other polypeptide;            [74] the polypeptide of any one of [1] to [73], wherein the            polypeptide is an antigen-binding molecule;            [75] the polypeptide of [74], wherein the antigen-binding            molecule is an antibody, a bispecific antibody, or an Fc            fusion molecule such as a peptide Fc fusion protein or            scaffold Fc fusion protein;            [76] a pharmaceutical composition comprising the polypeptide            of [74] or [75] and a medically acceptable carrier;            [77] a method for altering the function of a polypeptide            comprising an Fc region, which comprises the steps of            heterodimerizing the Fc region by introducing an amino acid            mutation into the first polypeptide and/or the second            polypeptide constituting the Fc region, and introducing an            amino acid mutation to alter the Fc region function compared            to when the Fc region forms a homodimer; and            [78] a method for producing a polypeptide comprising an Fc            region, which comprises the steps of heterodimerizing the Fc            region by introducing an amino acid mutation into the first            polypeptide and/or the second polypeptide constituting the            Fc region, and introducing an amino acid mutation to alter            the Fc region function compared to when the Fc region forms            a homodimer.

Effects of the Invention

The present invention provides polypeptides that are suitable aspharmaceuticals, wherein their binding activities and physicalproperties (for example, stability and homogeneity) have been improvedby altering the amino acid sequence of the antibody constant region.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of a complex of Fc region and FcRn. FcRnbinds to CH2 and CH3 of each antibody H chain, and is bound to the wholeantibody in a symmetric fashion.

FIG. 2 shows the structure of a complex of IgA and FcαR which is an IgAreceptor. FcαR binds to Cα2 and Cα3 of each IgA H chain, and is bound tothe whole antibody in a symmetric fashion.

FIG. 3 shows the structure of a complex of Fc region and FcγRIII. Forthe H chain, CH2, and CH3, those on the left side of the figure arereferred to as the H_(A) chain, CH_(A)2, and CH_(A)3, and those on theright side are referred to as the H_(B) chain, CH_(B)2, and CH_(B)3,respectively.

FIG. 4 shows detailed interactions between A327 in each H chain andFcγRIII. (A) shows the interaction between A327 in CH_(A)2 and FcγRIII.(B) shows the interaction between A327 in CH_(B)2HB and FcγRIII. InFcγRIII, the colors black, grey, and white indicate the basic portion,neutral portion, and acidic portion, respectively.

FIG. 5 shows comparison of FcγR-binding activities of antibodies intowhich D356K, H435R, and/or K439E have been introduced. The bindingactivity of GpH7-G1d/GpL16-k0 (SEQ ID NOs: 2 and 5) to each FcγR wasdefined as 100. The samples used for the evaluation and their sequenceswere GpH7-G1d/GpL16-k0 (SEQ ID NOs: 2 and 5), GpH7-A5/GpH7-B3/GpL16-k0(SEQ ID NOs: 3, 4, and 5), GpH7-B3/GpL16-k0 (SEQ ID NOs: 4 and 5), andGpH7-A5/GpL16-k0 (SEQ ID NO: 3 and 5).

FIG. 6 shows comparison of FcγR-binding activities of antibodies intowhich G237A has been introduced. The binding activity ofGpH7-G1d/GpL16-k0 (SEQ ID NOs: 2 and 5) to each FcγR was defined as 100.The samples used for the evaluation and their sequences wereGpH7-G1d/GpL16-k0 (SEQ ID NOs: 2 and 5), GpH7-A5/GpH7-B3/GpL16-k0 (SEQID NOs: 3, 4, and 5), GpH7-B3/GpL16-k0 (SEQ ID NOs: 4 and 5),GpH7-A5/GpL16-k0 (SEQ ID NOs: 3 and 5), GpH7-A26/GpH7-B3/GpL16-k0 (SEQID NOs: 6, 4, and 5), and GpH7-A26/GpL16-k0 (SEQ ID NOs: 6 and 5).

FIG. 7 shows comparison of FcγR-binding activities of homodimerizedantibodies and heterodimerized antibodies into which G237L has beenintroduced. The binding activity of GpH7-G1d/GpL16-k0 (SEQ ID NOs: 2 and5) to each FcγR was defined as 100. The samples used for the evaluationand their sequences were GpH7-G1d/GpL16-k0 (SEQ ID NOs: 2 and 5),GpH7-A5/GpH7-B3/GpL16-k0 (SEQ ID NOs: 3, 4, and 5), GpH7-B3/GpL16-k0(SEQ ID NOs: 4 and 5), GpH7-A5/GpL16-k0 (SEQ ID NO: 3 and 5),GpH7-A29/GpH7-B3/GpL16-k0 (SEQ ID NOs: 7, 4, and 5), andGpH7-A29/GpL16-k0 (SEQ ID NOs: 7 and 5).

FIG. 8 shows comparison of FcγR-binding activities of homodimerizedantibodies and heterodimerized antibodies into which L328E has beenintroduced. The binding activity of GpH7-G1d/GpL16-k0 (SEQ ID NOs: 2 and5) to each FcγR was defined as 100. The samples used for the evaluationand their sequences were GpH7-G1d/GpL16-k0 (SEQ ID NOs: 2 and 5),GpH7-A5/GpH7-B3/GpL16-k0 (SEQ ID NOs: 3, 4, and 5), GpH7-B3/GpL16-k0(SEQ ID NOs: 4 and 5), GpH7-A5/GpL16-k0 (SEQ ID NO: 3 and 5),GpH7-A42/GpH7-B3/GpL16-k0 (SEQ ID NOs: 8, 4, and 5), andGpH7-A42/GpL16-k0 (SEQ ID NOs: 8 and 5).

FIG. 9 shows comparison of FcγR-binding activities of homodimerizedantibodies and heterodimerized antibodies into which L328D has beenintroduced. The binding activity of GpH7-G1d/GpL16-k0 (SEQ ID NOs: 2 and5) to each FcγR was defined as 100. The samples used for the evaluationand their sequences were GpH7-G1d/GpL16-k0 (SEQ ID NOs: 2 and 5),GpH7-A5/GpH7-B3/GpL16-k0 (SEQ ID NOs: 3, 4, and 5), GpH7-B3/GpL16-k0(SEQ ID NOs: 4 and 5), GpH7-A5/GpL16-k0 (SEQ ID NO: 3 and 5),GpH7-A43/GpH7-B3/GpL16-k0 (SEQ ID NOs: 9, 4, and 5), andGpH7-A43/GpL16-k0 (SEQ ID NOs: 9 and 5).

FIG. 10 shows comparison of FcγR-binding activities of homodimerizedantibodies and heterodimerized antibodies into which L234E has beenintroduced. The binding activity of GpH7-G1d/GpL16-k0 (SEQ ID NOs: 2 and5) to each FcγR was defined as 100. The samples used for the evaluationand their sequences were GpH7-G1d/GpL16-k0 (SEQ ID NOs: 2 and 5),GpH7-A5/GpH7-B3/GpL16-k0 (SEQ ID NOs: 3, 4, and 5), GpH7-B3/GpL16-k0(SEQ ID NOs: 4 and 5), GpH7-A5/GpL16-k0 (SEQ ID NO: 3 and 5),GpH7-A5/GpH7-B16/GpL16-k0 (SEQ ID NOs: 3, 10, and 5), andGpH7-B16/GpL16-k0 (SEQ ID NOs: 10 and 5).

FIG. 11 shows comparison of FcγR-binding activities of homodimerizedantibodies and heterodimerized antibodies into which L234D has beenintroduced. The binding activity of GpH7-G1d/GpL16-k0 (SEQ ID NOs: 2 and5) to each FcγR was defined as 100. The samples used for the evaluationand their sequences were GpH7-G1d/GpL16-k0 (SEQ ID NOs: 2 and 5),GpH7-A5/GpH7-B3/GpL16-k0 (SEQ ID NOs: 3, 4, and 5), GpH7-B3/GpL16-k0(SEQ ID NOs: 4 and 5), GpH7-A5/GpL16-k0 (SEQ ID NO: 3 and 5),GpH7-A5/GpH7-B17/GpL16-k0 (SEQ ID NOs: 3, 11, and 5), andGpH7-B17/GpL16-k0 (SEQ ID NOs: 11 and 5).

FIG. 12 shows interactions of P329 in the Fc region with FcγRIII. Forthe H chain, CH2, and CH3, those on the left side of the figure arereferred to as the H_(A) chain, CH_(A)2, and CH_(A)3, and those on theright side are referred to as the H_(B) chain, CH_(B)2, and CH_(B)3,respectively. This figure shows that Pro at position 329 (EU numbering)in the Fc region interacts with FcγRIII mainly through CH_(A)2 which isa CH2 domain. For the H chain, CH2, and CH3, those on the left side ofthe figure are referred to as the H_(A) chain, CH_(A)2, and CH_(A)3, andthose on the right side are referred to as the H_(B) chain, CH_(B)2, andCH_(B)3, respectively.

FIG. 13 shows comparison of the effects on FcγR-binding activities ofhomodimerized antibodies and heterodimerized antibodies into whichP329R, P329K, P329D, or P329E has been introduced. The binding activityof GpH7-A5/GpH7-B3/GpL16-k0 (SEQ ID NOs: 3, 4, and 5) to each FcγR wasdefined as 100. The samples used for the evaluation and their sequenceswere GpH7-A5/GpH7-B3/GpL16-k0 (SEQ ID NOs: 3, 4, and 5),GpH7-A5/GpH7-B12/GpL16-k0 (SEQ ID NOs: 3, 12, and 5),GpH7-A5/GpH7-B13/GpL16-k0 (SEQ ID NOs: 3, 13, and 5),GpH7-A5/GpH7-B14/GpL16-k0 (SEQ ID NOs: 3, 14, and 5),GpH7-A5/GpH7-B15/GpL16-k0 (SEQ ID NOs: 3, 15, and 5), GpH7-B12/GpL16-k0(SEQ ID NOs: 12 and 5), GpH7-B13/GpL16-k0 (SEQ ID NOs: 13 and 5),GpH7-B14/GpL16-k0 (SEQ ID NOs: 14 and 5), and GpH7-B15/GpL16-k0 (SEQ IDNOs: 15 and 5).

FIG. 14 shows comparison of binding activities to each FcγR of aheterodimerized antibody with G237A introduced into one of the H chains,when P329R is introduced into the same H chain or into the other Hchain. The binding activity of GpH7-A5/GpH7-B3/GpL16-k0 (SEQ ID NOs: 3,4, and 5) to each FcγR was defined as 100. The samples used for theevaluation and their sequences were GpH7-A5/GpH7-B3/GpL16-k0 (SEQ IDNOs: 3, 4, and 5), GpH7-A5/GpH7-B12/GpL16-k0 (SEQ ID NOs: 3, 12, and 5),GpH7-A48/GpH7-B3/GpL16-k0 (SEQ ID NOs: 16, 4, and 5),GpH7-A26/GpH7-B3/GpL16-k0 (SEQ ID NOs: 6, 4, and 5),GpH7-A26/GpH7-B12/GpL16-k0 (SEQ ID NOs: 6, 12, and 5), andGpH7-A45/GpH7-B3/GpL16-k0 (SEQ ID NOs: 17, 4, and 5).

FIG. 15 shows comparison of binding activities to each FcγR of aheterodimerized antibody with L234D introduced into one of the H chains,when P329R is introduced into the same H chain or into the other Hchain. The binding activity of GpH7-A5/GpH7-B3/GpL16-k0 (SEQ ID NOs: 3,4, and 5) to each FcγR was defined as 100. The samples used for theevaluation and their sequences were GpH7-A5/GpH7-B3/GpL16-k0 (SEQ IDNOs: 3, 4, and 5), GpH7-A5/GpH7-B12/GpL16-k0 (SEQ ID NOs: 3, 12, and 5),GpH7-A48/GpH7-B3/GpL16-k0 (SEQ ID NOs: 16, 4, and 5),GpH7-A5/GpH7-B17/GpL16-k0 (SEQ ID NOs: 3, 11, and 5),GpH7-A48/GpH7-B17/GpL16-k0 (SEQ ID NOs: 16, 11, and 5), andGpH7-A5/GpH7-B41/GpL16-k0 (SEQ ID NOs: 3, 18, and 5).

FIG. 16 shows comparison of FcγRIa-binding activities of homodimerizedantibodies and heterodimerized antibodies into which identicalalterations have been introduced. The horizontal axis shows the Ho/Convalues, and the vertical axis shows the He/Co values. He/Con is a valueobtained by dividing the FcγRIa-binding activity of the heterodimerizedantibody GpH7-A5/GpH7-B3 variant/GpL16-k0, which uses a mutated GpH7-B3variant for one of the H chains, by the FcγRIa-binding activity of theheterodimerized antibody GpH7-A5/GpH7-B3/GpL16-k0 (SEQ ID NOs: 3, 4, and5), which uses the unmutated GpH7-B3, and multiplying the result by 100.Ho/Con is a value obtained by dividing the FcγRIa-binding activity ofthe homodimerized antibody GpH7-B3 variant/GpL16-k0, which uses amutated GpH7-B3 variant for both H chains, by the FcγRIa-bindingactivity of the homodimerized antibody GpH7-B3/GpL16-k0 (SEQ ID NOs: 4and 5), which uses the unmutated GpH7-B3, and multiplying the result by100.

FIG. 17 shows comparison of FcγRIIa R-binding activities ofhomodimerized antibodies and heterodimerized antibodies into whichidentical alterations have been introduced. The horizontal axis showsthe Ho/Con values, and the vertical axis shows the He/Co values. He/Conis a value obtained by dividing the FcγRIIa R-binding activity of theheterodimerized antibody GpH7-A5/GpH7-B3 variant/GpL16-k0, which uses amutated GpH7-B3 variant for one of the H chains, by the FcγRIIaR-binding activity of the heterodimerized antibodyGpH7-A5/GpH7-B3/GpL16-k0 (SEQ ID NOs: 3, 4, and 5), which uses theunmutated GpH7-B3. Ho/Con is a value obtained by dividing the FcγRIIaR-binding activity of the homodimerized antibody GpH7-B3variant/GpL16-k0, which uses a mutated GpH7-B3 variant for both Hchains, by the FcγRIIa R-binding activity of the homodimerized antibodyGpH7-B3/GpL16-k0 (SEQ ID NOs: 4 and 5), which uses the unmutatedGpH7-B3, and multiplying the result by 100.

FIG. 18 shows comparison of FcγRIIa H-binding activities ofhomodimerized antibodies and heterodimerized antibodies into whichidentical alterations have been introduced. The horizontal axis showsthe Ho/Con values, and the vertical axis shows the He/Co values. He/Conis a value obtained by dividing the FcγRIIa H-binding activity of theheterodimerized antibody GpH7-A5/GpH7-B3 variant/GpL16-k0, which uses amutated GpH7-B3 variant for one of the H chains, by the FcγRIIaH-binding activity of the heterodimerized antibodyGpH7-A5/GpH7-B3/GpL16-k0 (SEQ ID NOs: 3, 4, and 5), which uses theunmutated GpH7-B3. Ho/Con is a value obtained by dividing the FcγRIIaH-binding activity of the homodimerized antibody GpH7-B3variant/GpL16-k0, which uses a mutated GpH7-B3 variant for both Hchains, by the FcγRIIa H-binding activity of the homodimerized antibodyGpH7-B3/GpL16-k0 (SEQ ID NOs: 4 and 5), which uses the unmutatedGpH7-B3, and multiplying the result by 100.

FIG. 19 shows comparison of FcγRIIb-binding activities of homodimerizedantibodies and heterodimerized antibodies into which identicalalterations have been introduced. The horizontal axis shows the Ho/Convalues, and the vertical axis shows the He/Co values. He/Con is a valueobtained by dividing the FcγRIIb-binding activity of the heterodimerizedantibody GpH7-A5/GpH7-B3 variant/GpL16-k0, which uses a mutated GpH7-B3variant for one of the H chains, by the FcγRIIb-binding activity of theheterodimerized antibody GpH7-A5/GpH7-B3/GpL16-k0 (SEQ ID NOs: 3, 4, and5), which uses the unmutated GpH7-B3. Ho/Con is a value obtained bydividing the FcγRIIb-binding activity of the homodimerized antibodyGpH7-B3 variant/GpL16-k0, which uses a mutated GpH7-B3 variant for bothH chains, by the FcγRIIb-binding activity of the homodimerized antibodyGpH7-B3/GpL16-k0 (SEQ ID NOs: 4 and 5), which uses the unmutatedGpH7-B3, and multiplying the result by 100.

FIG. 20 shows comparison of FcγRIIIa-binding activities of homodimerizedantibodies and heterodimerized antibodies into which identicalalterations have been introduced. The horizontal axis shows the Ho/Convalues, and the vertical axis shows the He/Co values. The horizontalaxis shows the Ho/Con values, and the vertical axis shows the He/Covalues. He/Con is a value obtained by dividing the FcγRIIIa-bindingactivity of the heterodimerized antibody GpH7-A5/GpH7-B3variant/GpL16-k0, which uses a mutated GpH7-B3 variant for one of the Hchains, by the FcγRIIIa-binding activity of the heterodimerized antibodyGpH7-A5/GpH7-B3/GpL16-k0 (SEQ ID NOs: 3, 4, and 5), which uses theunmutated GpH7-B3. Ho/Con is a value obtained by dividing the FcγRIIIabinding activity of the homodimerized antibody GpH7-B3 variant/GpL16-k0,which uses a mutated GpH7-B3 variant for both H chains, by theFcγRIIIa-binding activity to of the homodimerized antibodyGpH7-B3/GpL16-k0 (SEQ ID NOs: 4 and 5), which uses the unmutatedGpH7-B3, and multiplying the result by 100.

FIG. 21 shows a conceptual diagram comparing FcγR-binding of each of thehomodimerized antibodies and heterodimerized antibodies which use Hchains into which alterations have been introduced. When a plotted pointis contained within Region i, it means that the alteration introducedinto the Fc region has the effect of yielding He/Con>100, Ho/Con<100,He/Con>Ho/Con. When a plotted point is contained within Region ii, itmeans that the alteration introduced into the Fc region has the effectof yielding He/Con>100, Ho/Con>100, He/Con>Ho/Con. When a plotted pointis contained within Region iii, it means that the alteration introducedinto the Fc region has the effect of yielding He/Con>100, Ho/Con>100,He/Con<Ho/Con.

FIG. 22 shows the number of combinations produced when three types ofalterations are introduced into any of the H chains of a heterodimerizedantibody. Each of the filled circles (●), filled triangle (▴), andfilled square (▪) refers to a different alteration.

FIG. 23 shows the interaction between FcγRIII and each of the residuesS239, A330, and I332 in the antibody Fc region. Cited from Proc. Natl.Acad. Sci. USA, 103, 4005-4010, 2006.

FIG. 24 shows comparison of binding activities to activating FcγR andinhibitory FcγR. It is a conceptual diagram comparing the bindingactivities of each variant to activating FcγR and inhibitory FcγR. Thevertical axis shows the activity of each variant towards activating FcγR(Activating Receptor), and the horizontal axis shows the bindingactivity of each variant to inhibitory FcγR (Inhibitory Receptor). Thebinding activities of the naturally-occurring antibody to activatingFcγR and inhibitory FcγR were each set to 100. When a variant hasenhanced binding activity to activating FcγR over thenaturally-occurring antibody and decreased binding activity toinhibitory FcγR, the antibody is plotted in Region a (shadow area). Whena variant has enhanced binding activity to inhibitory FcγR over thenaturally-occurring antibody, and decreased binding activity toactivating FcγR, the antibody is plotted in Region c (shaded area).

FIG. 25 shows comparison of binding activities to activating FcγR andinhibitory FcγR. It is a conceptual diagram comparing the bindingactivities of each variant to activating FcγR and inhibitory FcγR. Thevertical axis shows the binding activity of a naturally-occurringantibody to activating FcγR (Activating Receptor), and the horizontalaxis shows the binding activity to inhibitory FcγR (InhibitoryReceptor). The binding activities of the naturally-occurring antibody toactivating FcγR and inhibitory FcγR were each set to 100. When the valueobtained by dividing the binding activity of a variant to activatingFcγR by the binding activity to inhibitory FcγR is 1.2 or greater, theantibody is plotted in Region b (shadow area). When the value obtainedby dividing the binding activity of a variant to activating FcγR by thebinding activity to inhibitory FcγR is 0.8 or lower, the antibody isplotted in Region d (shaded area).

FIG. 26 shows comparison of binding activities of a heterodimerizedantibody to FcγRIa and FcγRIIb. It is a diagram that compares thebinding activities of an altered heterodimerized antibody to FcγRIawhich is an activating FcγR and to FcγRIIb which is an inhibitory FcγR.The horizontal axis shows the values of Ho/Con for inhibitory FcγR, andthe vertical axis shows the values for He/Con for activating FcγR.He/Con is a value obtained by dividing the FcγR-binding activity of theheterodimerized antibody GpH7-A5/GpH7-B3 variant/GpL16-k0 which has amutated Fc, by the FcγR-binding activity of the heterodimerized antibodyGpH7-A5/GpH7-B3/GpL16-k0 (SEQ ID NOs: 3, 4, and 5) which has nointroduction of alterations, and multiplying the result by 100.

FIG. 27 shows comparison of binding activities of a heterodimerizedantibody to FcγRIIa R and FcγRIIb. It is a diagram that compares thebinding activities of an altered heterodimerized antibody to FcγRIIa Rwhich is an activating FcγR and to FcγRIIb which is an inhibitory FcγR.The horizontal axis shows the values of He/Con for inhibitory FcγR, andthe vertical axis shows the values for He/Con for activating FcγR.He/Con is a value obtained by dividing the FcγR-binding activity of theheterodimerized antibody GpH7-A5/GpH7-B3 variant/GpL16-k0, which has amutated Fc, by the FcγR-binding activity of the heterodimerized antibodyGpH7-A5/GpH7-B3/GpL16-k0 (SEQ ID NOs: 3, 4, and 5) which has nointroduction of alterations, and multiplying the result by 100.

FIG. 28 shows comparison of binding activities of a heterodimerizedantibody to FcγRIIa H and FcγRIIb. It is a diagram that compares thebinding activities of an altered heterodimerized antibody to FcγRIIa Hwhich is an activating FcγR, and to FcγRIIb which is an inhibitory FcγR.The horizontal axis shows the values of He/Con for inhibitory FcγR, andthe vertical axis shows the values of He/Con for activating FcγR. He/Conis a value obtained by dividing the FcγR-binding activity of theheterodimerized antibody GpH7-A5/GpH7-B3 variant/GpL16-k0, which has amutated Fc, by the FcγR-binding activity of the heterodimerized antibodyGpH7-A5/GpH7-B3/GpL16-k0 (SEQ ID NOs: 3, 4, and 5) which has nointroduction of alterations, and multiplying the result by 100.

FIG. 29 shows comparison of binding activities of a heterodimerizedantibody to FcγRIIIa and FcγRIIb. It is a diagram that compares thebinding activities of an altered heterodimerized antibody to FcγRIIIawhich is an activating FcγR and to FcγRIIb which is an inhibitory FcγR.The horizontal axis shows the values of He/Con for inhibitory FcγR, andthe vertical axis shows the values of He/Con for activating FcγR. He/Conis a value obtained by dividing the FcγR-binding activity of theheterodimerized antibody GpH7-A5/GpH7-B3 variant/GpL16-k0 which has amutated Fc, by the FcγR-binding activity of the heterodimerized antibodyGpH7-A5/GpH7-B3/GpL16-k0 (SEQ ID NOs: 3, 4, and 5) which has nointroduction of alterations, and multiplying the result by 100.

FIG. 30 shows comparison of the effects of combinations of L234Y, G236Wand S298A, with S239D, A330L and I332E on the thermal stability ofantibodies. It is a graph that compares the changes in monomer contentafter a heat accelerated stability study (40° C. for two weeks or fourweeks) was performed for homodimerized antibodies and heterodimerizedantibodies with L234Y, G236W and S298A, homodimerized antibodies andheterodimerized antibodies with S239D, A330L, and I332E, andheterodimerized antibodies in which L234Y, G236W and S298A areintroduced into one of the H chains and S239D, A330L, and I332E areintroduced into the other H chain. The samples used for the evaluationand their sequences were GpH7-G1d/GpL16-k0 (SEQ ID NOs: 2 and 5),GpH7-A5/GpH7-B3/GpL16-k0 (SEQ ID NOs: 3, 4, and 5),GpH7-TA7/GpH7-B3/GpL16-k0 (SEQ ID NOs: 31, 4, and 5),GpH7-A5/GpH7-B78/GpL16-k0 (SEQ ID NO: 3, 41, and 5),GpH7-TA7/GpH7-TA45/GpL16-k0 (SEQ ID NOs: 31, 32, and 5),GpH7-A57/GpH7-B78/GpL16-k0 (SEQ ID NO: 40, 41, and 5), andGpH7-TA7/GpH7-B78/GpL16-k0 (SEQ ID NO: 31, 41, and 5).

FIG. 31 shows a result of examining the ADCC activities ofheterodimerized antibodies. The cell line used for the evaluation wasSK-pca13a, and the E/T ratio was 50. Human PBMCs were used as theeffector cell. The samples used for the evaluation and their sequenceswere GpH7-G1d/GpL16-k0 (SEQ ID NOs: 2 and 5), GpH7-A5/GpH7-B3/GpL16-k0(SEQ ID NOs: 3, 4, and 5), GpH7-A5/GpH7-B78/GpL16-k0 (SEQ ID NO: 3, 41,and 5), GpH7-TA7/GpH7-B3/GpL16-k0 (SEQ ID NOs: 31, 4, and 5),GpH7-A57/GpH7-B78/GpL16-k0 (SEQ ID NO: 40, 41, and 5),GpH7-TA7/GpH7-TA45/GpL16-k0 (SEQ ID NOs: 31, 32, and 5), andGpH7-TA7/GpH7-B78/GpL16-k0 (SEQ ID NOs: 31, 41, and 5). The verticalaxis shows the antibody cytotoxic activity, and the horizontal axisshows the antibody concentration (μg/mL).

FIG. 32 shows the amino acid residues constituting the Fc regions ofIgG1 (SEQ ID NO: 76), IgG2 (SEQ ID NO: 77), IgG3 (SEQ ID NO: 78), andIgG4 (SEQ ID NO: 79), and their relation to Kabat's EU numbering(herein, also referred to as EU INDEX).

FIG. 33 shows a result of examining the ADCC activities of the Fcheterodimerized antibodies described in Example 12. The cell line usedfor the evaluation was SK-pca13a, and the E/T ratio was 50. Human PBMCswere used as the effector cell. The samples used for the evaluation andtheir sequences were GpH7-G1d/GpL16-k0 (SEQ ID NOs: 2 and 5),GpH7-Kn033/GpH7-HI033/GpL16-k0 (SEQ ID NOs: 51, 56, and 5),GpH7-Kn032/GpH7-HI032/GpL16-k0 (SEQ ID NOs: 53, 58, and 5),GpH7-Kn045/GpH7-HI048/GpL16-k0 (SEQ ID NOs: 54, 59, and 5),GpH7-Kn056/GpH7-HI055/GpL16-k0 (SEQ ID NOs: 55, 60, and 5), andGpH7-Kn037/GpH7-HI036/GpL16-k0 (SEQ ID NOs: 52, 57, and 5). The verticalaxis shows the antibody cytotoxic activity, and the horizontal axisshows the antibody concentration (μg/mL).

FIG. 34 shows a result of examining the ADCC activity of theheterodimerized antibody H240-Kn061/H240-H1071/L73-k0. The cell lineused for the evaluation was SKE18, and the E/T ratio was 50. Human PBMCswere used as the effector cell. The samples used for the evaluation andtheir sequences were H240-Kn033/H240-H1033/L73-k0 (SEQ ID NOs: 84, 85,and 106), H240-Kn032/H240-H1032/L73-k0 (SEQ ID NOs: 86, 87, and 106),H240-Kn061/H240-H1071/L73-k0 (SEQ ID NOs: 81, 82, and 106), andH240-afucosyl_G1d (the amino acid sequence of H240-afucosyl_G1d is thesame as that of H240-G1d (SEQ ID NO: 83), but the fucose isremoved)/L73-k0 (SEQ ID NOs: 83 and 106). The vertical axis shows theantibody cytotoxic activity, and the horizontal axis shows the antibodyconcentration (μg/mL).

FIG. 35 shows the FcgRI-binding activity of a point mutant which usesthe heterodimerized antibody H240-Kn061/H240-H1071/L73-k0 as template.Relative KD on the vertical axis shows the value obtained by dividingthe KD (mol/L) of H240-Kn061/H240-H1071/L73-k0 to FcgRI by the KD ofeach variant. The numbers on the horizontal axis show the ranks when theRelative KDs are arranged in ascending order.

FIG. 36 shows the FcgRIIa R-binding activity of a point mutant whichuses the heterodimerized antibody H240-Kn061/H240-H1071/L73-k0 astemplate. Relative KD on the vertical axis shows the value obtained bydividing the KD (mol/L) of H240-Kn061/H240-H1071/L73-k0 to FcgRIIa R bythe KD of each variant. The numbers on the horizontal axis show theranks when the Relative KDs are arranged in ascending order.

FIG. 37 shows the FcgRIIa H-binding activity of a point mutant whichuses the heterodimerized antibody H240-Kn061/H240-H1071/L73-k0 astemplate. Relative KD on the vertical axis shows the value obtained bydividing the KD (mol/L) of H240-Kn061/H240-H1071/L73-k0 to FcgRIIa H bythe KD of each variant. The numbers on the horizontal axis show theranks when the Relative KDs are arranged in ascending order.

FIG. 38 shows the FcgRIIb-binding activity of a point mutant which usesthe heterodimerized antibody H240-Kn061/H240-H1071/L73-k0 as template.Relative KD on the vertical axis shows the value obtained by dividingthe KD (mol/L) of H240-Kn061/H240-H1071/L73-k0 to FcgRIIb by the KD ofeach variant. The numbers on the horizontal axis show the ranks when theRelative KDs are arranged in ascending order.

FIG. 39 shows the FcgRIIIa F-binding activity of a point mutant whichuses the heterodimerized antibody H240-Kn061/H240-H1071/L73-k0 astemplate. Relative KD on the vertical axis shows the value obtained bydividing the KD (mol/L) of H240-Kn061/H240-H1071/L73-k0 to FcgRIIIa F bythe KD of each variant. The numbers on the horizontal axis show theranks when the Relative KDs are arranged in ascending order.

FIG. 40 shows the FcgRIIIa V-binding activity of a point mutant whichuses the heterodimerized antibody H240-Kn061/H240-H1071/L73-k0 astemplate. Relative KD on the vertical axis shows the value obtained bydividing the KD (mol/L) of H240-Kn061/H240-H1071/L73-k0 to FcgRIIIa V bythe KD of each variant. The numbers on the horizontal axis show theranks when the Relative KDs are arranged in ascending order.

FIG. 41 shows a result of examining the ADCC activity of aheterodimerized antibody, H240-Kn072/H240-H1076/L73-k0. The cell lineused for the evaluation was MIAPaCa-2, and the E/T ratio was 25. HumanPBMCs were used as the effector cell. The samples used for theevaluation and their sequences were H240-Kn033/H240-H1033/L73-k0 (SEQ IDNOs: 84, 85, and 106), H240-Kn061/H240-H1071/L73-k0 (SEQ ID NOs: 81, 82,and 106), H240-afucosyl_G1d/L73-k0 (SEQ ID NOs: 83 and 106), andH240-Kn072/H240-H1076/L73-k0 (SEQ ID NOs: 90, 91, and 106). The verticalaxis shows antibody cytotoxic activity, and the horizontal axis showsantibody concentration (μg/mL).

FIG. 42 shows a result of examining the ADCC activity of an improvedantibody derived from the heterodimerized antibodyH240-Kn072/H240-H1076/L73-k0. The cell line used for the evaluation wasDLD-1, and the E/T ratio was 50. Human PBMCs were used as the effectorcell. The samples used for the evaluation and their sequences wereH240-Kn033/H240-H1033/L73-k0 (SEQ ID NOs: 84, 85, and 106),H240-afucosyl_G1d/L73-k0 (SEQ ID NOs: 83 and 106),H240-Kn113/H240-H1076/L73-k0 (SEQ ID NOs: 92, 91, and 106),H240-Kn115/H240-H1076/L73-k0 (SEQ ID NOs: 93, 91, and 106), andH240-Kn125/H240-H1076/L73-k0 (SEQ ID NOs: 94, 91, and 106). The verticalaxis shows antibody cytotoxic activity, and the horizontal axis showsantibody concentration (μg/mL).

FIG. 43 shows a result of examining the ADCC activity of aheterodimerized antibody, H240-Kn067/H240-H1068/L73-k0, and such. Thecell line used for the evaluation was DLD-1, and the E/T ratio was 50.Human PBMCs were used as the effector cell. The samples used for theevaluation and their sequences were H240-Kn033/H240-H1033/L73-k0 (SEQ IDNOs: 84, 85, and 106), H240-afucosyl_G1d/L73-k0 (SEQ ID NOs: 83 and106), H240-Kn067/H240-H1068/L73-k0 (SEQ ID NOs: 95, 96, and 106),H240-Kn120/H240-H1068/L73-k0 (SEQ ID NOs: 99, 96, and 106), andH240-Kn126/H240-H1068/L73-k0 (SEQ ID NOs: 100, 96, and 106). Thevertical axis shows antibody cytotoxic activity, and the horizontal axisshows antibody concentration (μg/mL).

FIG. 44 shows a crystal structure of Fc(WT)/FcgR2a R type (PDB ID=3RY6,J. Immunol. 2011, 187, 3208-321). The figure illustrates side chains ofGln 127, Leu132, and Phe160, which are three residues that differbetween FcgRIIa R type and FcgRIIb at the region near the interactinginterface between FcgRIIa R type and Fc in this structure. Thecorresponding amino acid residues in FcgRIIb are shown using one lettercodes in parentheses.

FIG. 45 shows an Fc (Kn120/H1068)/FcgRIIb extracellular domain complexwhose structure has been determined by X-ray crystal structure analysis.For the CH2 domain and the CH3 domain, those on the left are referred toas domain A and those on the right are referred to as domain B,respectively.

FIG. 46 shows a structure around Lys 127 (Gln in FcgRIIa R type) of theFcgRIIb extracellular region in the Fc (Kn120/H1068)/FcgRIIbextracellular region complex whose structure has been determined byX-ray crystal structure analysis. Since the electron density of the sidechain was not observed for Tyr296 of the Fc (Kn120/H1068), models werenot constructed for the side chain other than the Cα atom.

FIG. 47 shows a structure around Ser132 (Leu in FcgRIIa R type) of theFcgRIIb extracellular region in the Fc (Kn120/H1068)/FcgRIIbextracellular domain complex whose structure has been determined byX-ray crystal structure analysis. Since the electron density of the sidechain was not observed for D327 of the Fc (Kn120/H1068), models were notconstructed for the side chain other than the Cα atom.

FIG. 48 shows a structure around Tyr160 (Phe in FcgRIIa R type) of theFcgRIIb extracellular region in the Fc (Kn120/H1068)/FcgRIIbextracellular region complex whose structure has been determined byX-ray crystal structure analysis.

FIG. 49 shows an Fc (BP208)/FcgRIIb extracellular region complex whosestructure has been determined by X-ray crystal structure analysis. Forthe CH2 domain and the CH3 domain, those on the left are referred to asdomain A and those on the right are referred to as domain B,respectively.

FIG. 50 shows a comparison of CH2 domains A between the structure of theFc (BP208)/FcgRIIb extracellular region complex determined by X-raycrystal structure analysis and the structure of the Fc (WT)/FcgRIIaextracellular region complex (PDB code: 3RY6) determined by X-raycrystal structure analysis. In the figure, the structure of the Fc(BP208)/FcgRIIb extracellular region complex is depicted by a thickline, and the structure of the Fc (WT)/FcgRIIa extracellular regioncomplex is depicted by a thin line. CH2 domain A alone is illustrated inthe structure of the Fc (WT)/FcgRIIa extracellular region complex.

FIG. 51 shows a structure around Ser239 of Fc (BP208) CH2 domain B inthe Fc (BP208)/FcgRIIb extracellular region complex, which has beendetermined by X-ray crystal structure analysis.

FIG. 52 shows a structure around Ser239 of Fc (BP208) CH2 domain A inthe Fc (BP208)/FcgRIIb extracellular region complex, which has beendetermined by X-ray crystal structure analysis.

FIG. 53 shows results of analytical cation exchange chromatography for arepresentative altered antibody, H240-AK072/H240-BH076/L73-k0. A:H240-AK072/H240-BH076/L73-k0; B: H240-FA021/H240-FB084/L73-k0.

FIG. 54 shows results of cation exchange chromatography forfractionation (A) and analytical cation exchange rechromatography forthe collected fractions (B) of H240-FA021/H240-FB084/L73-k0 which is arepresentative altered antibody, H240-AK072/H240-BH076/L73-k0.

MODE FOR CARRYING OUT THE INVENTION

The following definitions are provided to facilitate understanding ofthe present invention described herein.

The present invention provides polypeptides comprising an Fc region,wherein the polypeptide is characterized in that the Fc region iscomposed of a heterodimer comprising a first polypeptide and a secondpolypeptide, wherein the polypeptide is characterized in that a functionof the Fc region is altered when compared to that of a polypeptidecharacterized in that the Fc region is composed of a homodimercomprising only the first polypeptide, and when compared to that of apolypeptide characterized in that the Fc region is composed of ahomodimer comprising only the second polypeptide. Furthermore, thepresent invention provides methods for producing the polypeptide, andmethods for altering functions of an Fc region-containing polypeptide.

In the present invention, the “polypeptide comprising an Fc region,wherein the polypeptide is characterized in that the Fc region iscomposed of a heterodimer comprising a first polypeptide and a secondpolypeptide” may be a polypeptide complex composed of a firstpolypeptide and a second polypeptide, as well as other multiplepolypeptides.

Herein, “first polypeptide” and “second polypeptide” mean polypeptidesconstituting an antibody Fc region. The terms “first polypeptide” and“second polypeptide” means that their sequences are different from eachother, and preferably at least the CH2 domain sequences are different.The polypeptides may be, for example, polypeptides that constitute theFc region of a naturally-occurring IgG, or polypeptides produced byaltering the polypeptides constituting the Fc region of anaturally-occurring IgG.

“Naturally-occurring IgGs” refers to polypeptides that belong to a classof antibodies practically encoded by immunoglobulin gamma genes andcomprise an amino acid sequence identical to those of IgGs found innature. For example, a naturally-occurring human IgG refers to anaturally-occurring human IgG1, naturally-occurring human IgG2,naturally-occurring human IgG3, naturally-occurring human IgG4, or such.Naturally-occurring IgGs also include mutants spontaneously producedfrom them.

“Polypeptides” of the present invention generally refers to peptides orproteins approximately ten amino acids or more in length. Furthermore,they are generally polypeptides derived from organisms, but are notparticularly limited, and for example, they may be polypeptidescomprising an artificially designed sequence. Furthermore, they may beany of naturally-occurring polypeptides, synthetic polypeptides,recombinant polypeptides, or such. Protein molecules of the presentinvention refer to molecules comprising the polypeptide.

Preferred examples of the polypeptides of the present invention includeantibodies. More preferred examples include naturally-occurring IgGs,particularly naturally-occurring human IgGs. “Naturally-occurring IgGs”refers to polypeptides belonging to a class of antibodies practicallyencoded by immunoglobulin gamma genes and comprising an amino acidsequence identical to those of IgGs found in nature. For example, anaturally-occurring human IgG means a naturally-occurring human IgG1,naturally-occurring human IgG2, naturally-occurring human IgG3,naturally-occurring human IgG4, or such. Naturally-occurring IgGs alsoinclude mutants spontaneously produced from them.

While an IgK (Kappa, κ chain), IgL1, IgL2, IgL3, IgL6, and IgL7 (Lambda,λ chain)-type constant region is present in the antibody light chainconstant region, it may be any light chain constant region. For thehuman IgK (Kappa) constant region and human IgL7 (Lambda) constantregion, a plurality of allotype sequences due to genetic polymorphismare described in “Sequences of proteins of immunological interest”, NIHPublication No. 91-3242, and any of them may be used in the presentinvention. Furthermore, in the present invention, a light chain constantregion may be a light chain constant region that has been altered withamino acid substitutions, additions, deletions, insertions, and/ormodifications or such. For the antibody Fc region, for example, Fcregions of the IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgMtypes exist. For example, a human IgG antibody Fc region can be used asthe antibody Fc region of the present invention, and human IgG1 antibodyFc regions are preferred. Fc regions that can be used as an Fc region ofthe present invention are, for example, those derived fromnaturally-occurring IgG constant regions, or specifically, a constantregion derived from naturally-occurring human IgG1 (SEQ ID NO: 76), aconstant region derived from naturally-occurring human IgG2 (SEQ ID NO:77), a constant region derived from naturally-occurring human IgG3 (SEQID NO: 78), and a constant region derived from naturally-occurring humanIgG4 (SEQ ID NO: 79). FIG. 32 shows the constant region sequences of thenaturally-occurring IgG1, IgG2, IgG3, and IgG4. Constant regions ofnaturally-occurring IgGs also include mutants spontaneously producedfrom them. For the constant regions of human IgG1, human IgG2, humanIgG3, and human IgG4 antibodies, a plurality of allotype sequences dueto genetic polymorphism are described in “Sequences of proteins ofimmunological interest”, NIH Publication No. 91-3242, and any of themmay be used in the present invention. In particular, for the human IgG1sequence, the amino acid sequence at positions 356 to 358 (EU numbering)may be either DEL or EEM.

Furthermore, the strength of interaction between an antibody Fc regionand FcγR has been reported to depend on Zn²⁺ ion concentration(Immunology Letters 143 (2012) 60-69). The antibody shows a strongerinteraction between the Fc region and FcgR when the Zn²⁺ ionconcentration of the Fc region is higher. Chelation of Zn²⁺ by His310and His435 present in CH3 of the antibody Fc region opens up each CH2domain of the Fc region at a distal position. This facilitatesinteraction between the CH2 domain and FcgR, and the interaction betweenthe Fc region and FcgR is enhanced. A non-limiting embodiment of the Fcregion of the present invention includes an Fc region in which His atposition 310, His at position 435, His at position 433, and/or Asn atposition 434 (EU numbering) is chelated with Zn²⁺.

In the present invention, “Fc region” refers to a region consisted of ahinge region or a part thereof, a CH2 domain, and a CH3 domain in anantibody molecule. According to EU numbering (herein, also called the EUINDEX), an IgG-class Fc region refers to, for example, a region fromcysteine at position 226 to the C terminus, or from proline at position230 to the C terminus, but is not limited thereto.

The Fc region may be obtained preferably by re-eluting fractionsadsorbed onto a protein A column or protein G column after partiallydigesting IgG1, IgG2, IgG3, IgG4 monoclonal antibodies or such using aprotease such as pepsin. The protease is not particularly limited aslong as it can digest a full-length antibody so as to produce Fab andF(ab′)2 in a restrictive manner by appropriately setting the enzymereaction conditions such as pH; and examples include pepsin and papain.

Herein, “heterodimerization” means constituting a single polypeptidefrom two polypeptides with different amino acid sequences, and“heterodimer” means a polypeptide composed of two polypeptides withdifferent amino acid sequences. Furthermore, “homodimerization” meansconstituting a single polypeptide from two polypeptides having the sameamino acid sequences, and “homodimer” means a polypeptide composed oftwo polypeptides having the same amino acid sequences; or a polypeptidecomposed of polypeptides having the same amino acid sequences excludingalterations made for the purpose of efficient heterodimerization oralterations made for the purpose of efficient purification ofheterodimers; or a polypeptide composed of polypeptides comprising thesame amino acids excluding alterations that have not been made for thepurpose of improving Fc function. In the present invention,“heterodimer” or “homodimer” preferably means “heterodimerization” or“homodimerization” for the Fc region, or preferably means“heterodimerization” or “homodimerization” for CH2 in the Fc region.Furthermore, “parent polypeptide” means a polypeptide beforeintroduction of alterations such as amino acid mutations.

The amino acid mutation of the present invention may be used alone or incombination with multiple amino acid mutations.

When multiple amino acid mutations are used in combination, the numberof mutations combined is not particularly limited, and can be setappropriately within a range that can achieve the objectives of theinvention, and examples include two or more to 30 or less, andpreferably two or more to 15 or less.

When multiple amino acid mutations are combined, the amino acidmutations may be introduced into only one of the two polypeptidesconstituting the Fc region, or they may be appropriately distributed toboth of the two polypeptides.

Furthermore, in the present invention, to achieve a higherfunction-modifying effect in the Fc region, it is preferable tointroduce at least one amino acid mutation that improves the Fc regionfunction when the mutation is introduced into only one of thepolypeptides, compared to when no mutation is introduced and when themutation is introduced into both Fc regions of the two polypeptides.

The site to be altered is not particularly limited as long as it is inan Fc region, and it can be appropriately set within a range that canachieve the objectives of the present invention; and examples includethe hinge region, CH2 region, and CH3 region.

More preferably, the site to be altered is a CH2 domain. CH2 domainrefers to positions 231 to 340 (EU numbering), and CH3 domain refers topositions 341 to 447 (EU numbering). For example, when introducingmutations into the amino acid sequence of a constant region derived fromhuman IgG1, amino acid residues at one or more positions selected fromamong 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172,173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200,201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214,215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228,229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242,243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256,257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270,271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284,285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298,299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312,313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326,327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340,341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354,355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368,369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382,383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396,397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410,411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424,425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438,439, 440, 441, 442, 443, 444, 445, 446, and 447 (EU numbering) may bealtered.

More specifically, when introducing alterations into the amino acidsequence of a human IgG1 constant region, amino acid residues at one ormore positions selected from among 226, 227, 228, 229, 230, 231, 232,233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246,247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260,261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274,275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288,289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302,303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316,317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330,331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344,345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358,359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372,373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386,387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400,401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414,415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428,429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442,443, 444, 445, 446, and 447 (EU numbering) may be altered.

More specifically, when introducing alterations into the amino acidsequence of a human IgG1 constant region, amino acid residues at one ormore positions selected from among 226, 227, 228, 229, 230, 231, 232,233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246,247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260,261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274,275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288,289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302,303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316,317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330,331, 332, 333, 334, 335, 336, 337, 338, 339, and 340 (EU numbering) maybe altered.

More specifically, when introducing alterations into the amino acidsequence of a human IgG1 constant region, amino acid residues at one ormore positions selected from among 231, 232, 233, 234, 235, 236, 237,238, 239, 240, 265, 266, 267, 268, 269, 270, 271, 295, 296, 298, 300,324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, and 337(EU numbering) may be altered.

In the present invention, amino acid alteration means any ofsubstitution, deletion, addition, insertion, and modification, or acombination thereof. In the present invention, amino acid alteration maybe rephrased as amino acid mutation, and they are used synonymously.

Substitution

When substituting amino acid residues, substitution to a different aminoacid residue is carried out with the objective of altering aspects suchas (a)-(c) described below:

(a) polypeptide backbone structure in the sheet-structure orhelical-structure region;

(b) electric charge or hydrophobicity at the target site; or

(c) size of the side chain.

Amino acid residues are classified into the following groups based ontheir general side chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, and ile;

(2) neutral hydrophilic: cys, ser, thr, asn, and gln;

(3) acidic: asp and glu;

(4) basic: his, lys, and arg;

(5) residues that affect the chain orientation: gly and pro; and

(6) aromatic: tip, tyr, and phe.

Substitution between amino acid residues within each of these amino acidgroups is referred to as conservative substitution, and amino acidresidue substitution between different groups is referred to asnon-conservative substitution.

Substitutions in the present invention may be conservative substitutionsor non-conservative substitutions, or a combination of conservativesubstitutions and non-conservative substitutions.

In addition to the amino acid mutations introduced based on the presentinvention, polypeptides of the present invention may further compriseadditional alterations. For example, additional alterations may beselected from any of amino acid substitutions, deletions, andmodifications, or combinations thereof.

For example, polypeptides of the present invention can be arbitrarilyaltered within a range that does not practically change the intendedfunction of the polypeptide. When the polypeptide of the presentinvention is an antibody, its heavy chain and light chain can bealtered. For example, such mutations can be performed throughconservative substitution of amino acid residues. Furthermore, even ifan alteration changes the intended function of a polypeptide of thepresent invention, the alteration may also be carried out as long as thechange in the function is in a range within the objectives of thepresent invention.

Amino acid sequence alteration of the present invention includespost-translational modification. A specific post-translationalmodification may be addition or deletion of a sugar chain. For example,in the IgG1 constant region, the amino acid residue at position 297 (EUnumbering) may be sugar chain-modified. The sugar-chain structure forthe modification is not limited. Generally, antibodies expressed ineukaryotic cells comprise glycosylation in the constant region.Therefore, antibodies expressed in cells such as those below arenormally modified by some type of sugar chain:

-   -   antibody-producing cells of mammals    -   eukaryotic cells transformed with an expression vector        comprising a DNA encoding an antibody

Eukaryotic cells shown here include yeast and animal cells. For example,CHO cells and HEK293H cells are representative animal cells used intransformation with an expression vector comprising an antibody-encodingDNA. On the other hand, those without glycosylation at this site arealso included in the antibody of the present invention. Antibodies whoseconstant region is not glycosylated can be obtained by expressing anantibody-encoding gene in prokaryotic cells such as Escherichia coli.

In the present invention, additional alterations specifically include,for example, addition of sialic acid to the sugar chain of an Fc region(MAbs. 2010 September-October; 2(5): 519-27).

When the polypeptide of the present invention is an antibody, forexample, amino acid substitutions that improve FcRn-binding activity (J.Immunol. 2006 Jan. 1; 176(1): 346-56; J Biol Chem. 2006 Aug. 18;281(33): 23514-24; Int. Immunol. 2006 December; 18(12): 1759-69; NatBiotechnol. 2010 February; 28(2): 157-9; WO/2006/019447; WO/2006/053301;and WO/2009/086320), and amino acid substitutions for improving antibodyheterogeneity or stability (WO/2009/041613) may be introduced into anantibody constant region portion.

To produce a heterodimerized polypeptide of the present invention, it isrequired that polypeptides having amino acids that differ from eachother are associated, or a heterodimerized polypeptide of interest isseparated from other homodimerized polypeptides.

For association of polypeptides having different amino acids from eachanother and comprising an Fc region, a technique of suppressingunintended association between H chains by introducing electrostaticrepulsion into the interface of the second constant region of theantibody H chain (CH2) or the third constant region of the H chain (CH3)(WO 2006/106905) can be applied.

In the technology of suppressing unintended association between H chainsby introducing electrostatic repulsion into the interface of CH2 or CH3,examples of amino acid residues in contact at the interface of otherconstant regions of the H chain include the residue at position 356 (EUnumbering), the residue at position 439 (EU numbering), the regionfacing the residue at position 357 (EU numbering), the residue atposition 370 (EU numbering), the residue at position 399 (EU numbering),and the residue at position 409 (EU numbering) in the CH3 domain.

More specifically, for example, in an antibody containing two types of Hchain CH3 domains, the antibody in which one to three pairs of aminoacid residues selected from the amino acid residues shown below in (1)to (3) in the first H chain CH3 domain have the same type of charge canbe produced:

-   (1) amino acid residues at positions 356 and 439 (EU numbering)    which are amino acid residues contained in the H chain CH3 domain;-   (2) amino acid residues at positions 357 and 370 (EU numbering)    which are amino acid residues contained in the H chain CH3 domain;    and-   (3) amino acid residues at positions 399 and 409 (EU numbering)    which are amino acid residues contained in the H chain CH3 domain.

Furthermore, an antibody can be produced in which one to three pairs ofamino acid residues corresponding to the amino acid residue pairsindicated above in (1) to (3) having the same type of charge in thefirst H chain CH3 domain have charges opposite to the correspondingamino acid residues in the aforementioned first H chain CH3 domain,wherein the amino acid residue pairs are selected from the amino acidresidue pairs indicated above in (1) to (3) in the second H chain CH3domain which differs from the first H chain CH3 domain.

The respective amino acid residues of (1) to (3) mentioned above arepositioned close to each other when associated. Those skilled in the artcan find sites that correspond to the above-mentioned amino acidresidues of (1) to (3) by homology modeling and such using commerciallyavailable software for the desired H chain CH3 domain or H chainconstant region, and amino acid residues of these sites can be alteredwhen appropriate.

In the above-mentioned antibodies, for example, “charged amino acidresidues” are preferably selected from amino acid residues included ineither of groups (X) or (Y) below:

(X) glutamic acid (E) and aspartic acid (D); and

(Y) lysine (K), arginine (R), and histidine (H).

In the above-mentioned antibodies, the phrase “having the same type ofcharge” means that, for example, all of the two or more amino acidresidues are amino acid residues included in either of theabove-mentioned groups (X) and (Y). The phrase “having the oppositecharge” means that, for example, when at least one of the two or moreamino acid residues is an amino acid residue included in either one ofthe above-mentioned groups (X) and (Y), the remaining amino acidresidues are amino acid residues included in the other group.

In a preferred embodiment of the above-mentioned antibody, the first Hchain CH3 domain and the second H chain CH3 domain may be cross-linkedby disulfide bonds.

In the present invention, the amino acid residues to be altered are notlimited to amino acid residues of the antibody constant region orantibody variable region described above. Those skilled in the art canfind amino acid residues that form the interface in polypeptide mutantsor heteromultimers through homology modeling and such using commerciallyavailable software, and can alter the amino acid residues at those sitesto regulate association.

Other known technologies can also be used for the association ofpolypeptides of the present invention having different amino acids andcomprising an Fc region. Polypeptides having different amino acids andcomprising an Fc region can be efficiently associated with each other bysubstituting an amino acid side chain present in one of the H chainvariable regions of the antibody with a larger side chain (knob), andsubstituting an amino acid side chain present in the opposing variableregion of the other H chain with a smaller side chain (hole), to allowplacement of the knob within the hole (WO 1996/027011; and Ridgway J Bet al., Protein Engineering (1996) 9, 617-621; Merchant A M et al.Nature Biotechnology (1998) 16, 677-681).

In addition, other known technologies can also be used for theassociation of polypeptides having different amino acids and comprisingan Fc region. Association of polypeptides having different sequences canbe induced efficiently by complementary association of CH3, by using astrand-exchange engineered domain CH3 produced by changing part of theCH3 in one of the H chains of an antibody into an IgA-derived sequencecorresponding to that portion, and introducing a correspondingIgA-derived sequence into the complementary portion of the CH3 on theother H chain (Protein Engineering Design & Selection, 23; 195-202,2010). This known technology can also be used to efficiently induceassociation of polypeptides having different amino acids and comprisingan Fc region.

In addition, one can also use technologies for heterodimerized antibodyproduction using association of antibody CH1 and CL, and association ofVH and VL, which are described in WO 2011/028952.

Furthermore, even in cases where heterodimerized polypeptides cannot beformed efficiently, heterodimerized polypeptides can be obtained byseparating and purifying them from homodimerized polypeptides. Whenproducing a heterodimerized polypeptide consisting of a firstpolypeptide and a second polypeptide which have different sequences fromeach other, homodimerized polypeptides consisting of only two firstpolypeptides, and homodimerized polypeptide consisting of only twosecond polypeptide are mixed in as impurities. Known technologies can beused as a method for efficiently removing these two types ofhomodimerized polypeptides. A method has been reported to be able topurify two types of homodimers and the heterodimerized antibody ofinterest by ion exchange chromatography, by creating a difference inisoelectric points by introducing amino acid substitutions into thevariable regions of the two types of H chains (WO 2007114325). To date,as a method for purifying heterodimerized antibodies, a method usingProtein A has been reported to purify a heterodimerized antibodycomprising a mouse IgG2a H chain that binds to Protein A and a rat IgG2bH chain that does not bind to Protein A (WO 98050431 and WO 95033844).

Furthermore, a heterodimerized antibody alone can be efficientlypurified by using H chains in which amino acid residues at theIgG-Protein A binding site, positions 435 and 436 (EU numbering), aresubstituted with amino acids yielding different Protein A affinitiessuch as Tyr or His to change interaction of each of the H chains withProtein A, and using a Protein A column.

A plurality of these substitutions and technologies, for example, two ormore of them can be used in combination. Furthermore, these alterationscan be made separately to the first polypeptide and the secondpolypeptide when necessary. Polypeptides of the present invention mayalso be those produced based on the products of the above-mentionedalterations.

Amino acid sequence can be altered by various methods known to thoseskilled in the art. Such methods include the site-directed mutagenesismethod (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Nakagawa, M.(1995) An oligodeoxyribonucleotide-directed dual amber method forsite-directed mutagenesis. Gene 152: 271-275; Zoller, M J, and Smith, M.(1983) Oligonucleotide-directed mutagenesis of DNA fragments cloned intoM13 vectors. Methods Enzymol. 100: 468-500; Kramer, W, Drutsa, V,Jansen, H W, Kramer, B, Pflugfelder, M, and Fritz, H J (1984) The gappedduplex DNA approach to oligonucleotide-directed mutation construction.Nucleic Acids Res. 12: 9441-9456; Kramer W, and Fritz H J (1987)Oligonucleotide-directed construction of mutations via gapped duplex DNAMethods. Enzymol. 154, 350-367; and Kunkel, T A (1985) Rapid andefficient site-specific mutagenesis without phenotypic selection. ProcNatl Acad Sci USA. 82: 488-492), the PCR mutation method, and thecassette mutation method, but are not limited thereto.

In the present invention, the phrase “Fc region function” refers to, forexample, Fcγ receptor-binding activity of an Fc region (enhancement ofbinding activity or reduction of binding activity), selectivity of an Fcregion for Fcγ receptor isoform (improvement of binding selectivity),physicochemical stability of an Fc region, ADCC activity, and ADCPactivity. Here, Fcγ receptor isoform selectivity of an Fc region meansselective binding to specific isoforms of Fcγ receptor. Physicochemicalstability of an Fc region means, for example, an Fc region's thermalstability, stability to protease, stability to chemical treatment,stability to freeze thawing, storage stability, stability under acidicconditions, photostability, stability to shaking- orconcentration-associated stress, or maintenance of solubility in a widerange of solution conditions. Furthermore, an Fc region function mayrefer to a function combining two or more functions from Fcγreceptor-binding activity of an Fc region, Fcγ receptor isoformselectivity of an Fc region, and physicochemical stability of an Fcregion; and this means, for example, a function combining Fcγreceptor-binding activity of an Fc region and Fcγ receptor isoformselectivity of an Fc region, a function combining Fcγ receptor-bindingactivity of an Fc region and physicochemical stability of an Fc region,a function combining Fcγ receptor isoform selectivity of an Fc regionand physicochemical stability of the Fc region, and a function combiningFcγ receptor-binding activity of an Fc region, Fcγ receptor isoformselectivity of an Fc region, and physicochemical stability of an Fcregion.

In the present invention, “alteration of Fc region function” means, forexample, enhancement, reduction, or such of Fcγ receptor-bindingactivity of an Fc region when the Fc region function refers to the Fcγreceptor-binding activity of the Fc region. Improvement of selectivitymeans, for example, enhancing binding activity to an Fcγ receptor and atthe same time maintaining or lowering binding activities to other Fcγreceptors. Alternatively, improvement of selectivity means, for example,lowering binding activity to certain Fcγ receptors while maintaining orenhancing binding activities to other Fcγ receptors. Furthermore, forexample, when the Fc region function refers to Fcγ receptor subtypeselectivity of an Fc region, “alteration of Fc region function” meansimproving or lowering the Fcγ receptor subtype selectivity of the Fcregion. Alternatively, for example, when the Fc region function refersto physicochemical stability of an Fc region, “alteration of Fc regionfunction” means improving or lowering the physicochemical stability ofthe Fc region, suppressing a decrease in stability, or such; and morespecifically it means, for example, improving or lowering the Tm valueof the CH2 domain, suppressing a decrease in the Tm value, or such.

For example, improvement of combined functions of Fcγ receptor-bindingactivity of an Fc region, Fcγ receptor isoform selectivity of an Fcregion, and physicochemical stability of an Fc region does notnecessarily have to be improvement in every one of the Fcγreceptor-binding activity of an Fc region, the Fcγ receptor isoformselectivity of an Fc region, and the physicochemical stability of an Fcregion when compared to a control, and the improvement is acceptable aslong as the Fc region function is improved as a whole. Conversely, forexample, lowering of combined functions of the Fcγ receptor-bindingactivity of an Fc region, the Fcγ receptor isoform selectivity of an Fcregion, and the physicochemical stability of an Fc region does notnecessarily have to be decrease in every one of the Fcγ receptor-bindingactivity of an Fc region, the Fcγ receptor isoform selectivity of an Fcregion, and the physicochemical stability of an Fc region when comparedto a control, and the decrease is acceptable as long as the Fc regionfunction is lowered as a whole.

In the present invention, “Fcγ receptors” (herein, referred to as Fcγreceptors, FcγR, or FcgR) refers to receptors that may bind to the Fcregion of IgG1, IgG2, IgG3, and IgG4, and practically means any memberof the family of proteins encoded by the Fcγ receptor genes. In humans,this family includes FcγRI (CD64) including isoforms FcγRIa, FcγRIb, andFcγRIc; FcγRII (CD32) including isoforms FcγRIIa (including allotypesH131 (type H) and R131 (type R)), FcγRIIb (including FcγRIIb-1 andFcγRIIb-2), and FcγRIIc; and FcγRIII (CD16) including isoforms FcγRIIIa(including allotypes V158 and F158), and FcγRIIIb (including allotypesFcγRIIIb-NA1 and FcγRIIIb-NA2), and any human FcγRs, FcγR isoforms orallotypes yet to be discovered, but is not limited thereto. The FcγRsinclude human, mouse, rat, rabbit, and monkey-derived FcγRs but is notlimited thereto, and may be derived from any organism. Mouse FcγRsinclude FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2(CD16-2 or FcγRIV), and any mouse FcγRs, or FcγR isoforms or allotypesyet to be discovered, but are not limited thereto. Favorable examples ofsuch Fcγ receptors include human FcγRI (CD64), FcγRIIa (CD32), FcγRIIb(CD32), FcγRIIIa (CD16), and/or FcγRIIIb (CD16).

For FcγRs, there are activating receptors carrying the immunoreceptortyrosine-based activation motif (ITAM) and inhibitory receptor carryingthe immunoreceptor tyrosine-based inhibitory motif (ITIM). FcγRs areclassified into activating FcγRs: FcγRI, FcγRIIa R, FcγRII H, FcγRIIIa,and FcγRIIIb, and inhibitory FcγR: FcγRIIb.

The polynucleotide sequence and amino acid sequence of FcγRI are setforth in NM_000566.3 and NP_000557.1, respectively;

the polynucleotide sequence and amino acid sequence of FcγRIIa are setforth in BC020823.1 and AAH20823.1, respectively;

the polynucleotide sequence and amino acid sequence of FcγRIIb are setforth in BC146678.1 and AAI46679.1, respectively;

the polynucleotide sequence and amino acid sequence of FcγRIIIa are setforth in BC033678.1 and AAH33678.1, respectively; and

the polynucleotide sequence and amino acid sequence of FcγRIIIb are setforth in BC128562.1 and AAI28563.1, respectively (the RefSeqRegistration number).

In FcγRIIa, there are two allotypes, one where the amino acid atposition 131 of FcγRIIa is histidine (type H) and the other where thisamino acid is substituted with arginine (type R) (J. Exp. Med, 172:19-25, 1990). In FcγRIIb, there are two allotypes, one where the aminoacid at position 232 of FcγRIIb is isoleucine (type I) and the otherwhere this amino acid is substituted with threonine (type T) (Arthritis.Rheum. 46: 1242-1254 (2002)). In FcγRIIIa, there are two allotypes, onewhere the amino acid at position 158 of FcγRIIIa is valine (type V) andthe other where this amino acid is substituted with phenylalanine (typeF) (J. Clin. Invest. 100(5): 1059-1070 (1997)). Furthermore, inFcγRIIIb, there are two allotypes, the NA1 type and the NA2 type (J.Clin. Invest. 85: 1287-1295 (1990)).

One of the substances (the ligand) in observation of an interaction isimmobilized onto a gold thin film on a sensor chip, and by shining lightfrom the reverse side of the sensor chip so that total reflection takesplace at the interface between the gold thin film and glass, a portionof reduced reflection intensity is formed in part of the reflected light(SPR signal). When the other one of the substances (the analyte) inobservation of an interaction is made to flow on the sensor chip surfaceand the ligand binds to the analyte, the mass of the immobilized ligandmolecule increases and the refractive index of the solvent on the sensorchip surface changes. The position of the SPR signal shifts as a resultof this change in refractive index (on the other hand, the signalposition returns when this binding dissociates). The Biacore systemindicates the amount of shift mentioned above, or more specifically thetime variable of mass by plotting the change in mass on the sensor chipsurface on the ordinate as the measurement data (sensorgram). The amountof analyte bound to the ligand captured on the sensor chip surface isdetermined from the sensorgram (amount of change in the response on thesensorgram before and after interaction with the analyte). However,since the amount of binding also depends on the amount of ligand, it isnecessary to make the comparison under conditions where the amount ofligand is considered to be practically the same. Kinetic parameters suchas association rate constants (ka) and dissociation rate constants (kd)are determined from the curve of the sensorgram, and the affinities (KD)are determined from the ratio of these constants. In the BIACORE method,a method for measuring inhibition is preferably used. An example of themethod for measuring inhibition is described in Proc. Natl. Acad. SciUSA (2006) 103 (11), 4005-4010.

In the present invention, whether or not the binding activity towardseach type of Fcγ receptor is enhanced or decreased in a polypeptide orin an Fc region of the present invention can be determined, for example,by using Biacore (GE Healthcare) which is an interaction analyzer thatutilizes the surface plasmon resonance (SPR) phenomena, as shown in theExamples. Biacore includes any models such as Biacore T100, T200, X100,A100, 4000, 3000, 2000, 1000, and C. For the sensor chip, anysensorchips for Biacore such as CM7, CM5, CM4, CM3, C1, SA, NTA, L1,HPA, and Au chip can be used. For the running buffer, besides HBE-EP+, abuffer produced by using HEPES, phosphoric acid, ACES, Tris, citricacid, and such to adjust the pH to a near neutral pH such as 7.4 can beused. The measurements can be performed in the range of 4° C. to 37° C.A protein for capturing antibodies such as Protein A, Protein G, orProtein L, which captures an antibody, anti-human IgG antibody,anti-human IgG-Fab, anti-human L chain antibody, anti-human Fc antibody,antigen protein, or antigen peptide is immobilized onto a sensor chip bycoupling methods such as amine coupling, disulfide coupling, or aldehydecoupling. Various types of Fcγ receptors such as Fcγ receptor I, IIa Rtype, Ha H type, IIb, IIIc F type, V type, and IIIb are applied asanalytes, and interactions were measured to obtain sensorgrams. At thispoint, measurements can be carried out by adjusting the Fcγ receptorconcentration to a level in the range of several uM to several pMaccording to the strength of interaction such as KD of the sample to bemeasured. The dissociation constant (KD) is obtained based on themeasurement, and by observing whether the KD value is decreased orincreased, one can determine whether the binding activity of an Fcregion or a polypeptide of the present invention to various Fcγreceptors is increased or decreased. When capturing is carried out bythe antibody-capturing protein immobilized onto the sensor chip, thelevel of change in the sensorgram values before and after applyingvarious Fcγ receptors as analytes for the antibodies on the sensor chipis used as an indicator; and according to the degree of the valueincrease, one can determine whether the binding activity of thepolypeptide or Fc region of the present invention to various Fcγreceptors is increased or decreased. Alternatively, it is also possibleto immobilize various Fcγ receptors, instead of antibodies, onto thesensor chips, and make them interact with an antibody sample to beevaluated. Whether the binding activity of the Fc region or thepolypeptide of the present invention to the various Fcγ receptors isenhanced or decreased can be determined from the decrease or increase inKD values calculated from the interaction sensorgrams, or from thedegree of increase in the sensorgrams before and after allowing theantibody sample to react.

Specifically, the binding activity of an Fc region towards an Fcγreceptor can be measured by the Amplified Luminescent ProximityHomogeneous Assay (ALPHA) screening, the BIACORE method which utilizesthe surface plasmon resonance (SPR) phenomena, or such, in addition toELISA or fluorescence activated cell sorting (FACS) (Proc. Natl. Acad.Sci. USA (2006) 103 (11): 4005-4010).

ALPHA screening is performed by ALPHA technology which uses two beads, adonor and an acceptor, based on the following principles. Luminescentsignals are detected only when molecules bound to donor beads physicallyinteract with molecules bound to the acceptor beads, and the two beadsare in close proximity to each other. Laser-excited photosensitizer inthe donor beads converts ambient oxygen to excited-state singlet oxygen.Singlet oxygen is dispersed around the donor beads, and when it reachesthe adjacent acceptor beads, chemiluminescent reaction is induced in thebeads, and light is ultimately emitted. When the molecules bound to thedonor beads do not interact with the molecules bound to the acceptorbeads, the chemiluminescent reaction does not take place because singletoxygen produced by the donor beads does not reach the acceptor beads.

For example, a biotinylated test polypeptide is bound to streptavidin onthe donor beads, and Fcγ receptor tagged with glutathione S transferase(GST) is linked to the acceptor beads. In the absence of a competingpolypeptide, the test polypeptide interacts with the Fcγ receptor andproduces 520-620 nm signals. An untagged polypeptide competes with thetest polypeptide for interaction with the Fcγ receptor. Relative bindingactivities can be determined by quantifying the decrease in fluorescenceobserved as a result of the competition. Biotinylation of polypeptideusing Sulfo-NHS-biotin and such is well known. The method of expressingthe Fcγ receptor and GST in a cell carrying a fusion gene produced byfusing a polynucleotide encoding the Fcγ receptor in frame with apolynucleotide encoding GST in an expressible vector, and performingpurification using a glutathione column is appropriately adopted as amethod for tagging an Fcγ receptor with GST. The obtained signals arepreferably analyzed, for example, by fitting them to a one-sitecompetition model which uses a non-linear regression analysis usingsoftware such as GRAPHPAD PRISM (GraphPad, San Diego).

Tagging is not limited to the use of GST, and any tag such as histidinetag, MBP, CBP, Flag tag, HA tag, V5 tag, and c-myc tag, may be used.Furthermore, the binding of a test polypeptide to donor beads is notlimited to binding using the biotin-streptavidin reaction. Inparticular, when a test polypeptide is an antibody or peptide carryingFc such as an Fc fusion polypeptide, a method of binding a testpolypeptide through an Fc-recognizing protein such as Protein A orProtein G on the donor beads can be considered.

Reduction of binding to FcγR or FcγR-binding activity refers to bindingto FcγR with a substantially weaker binding activity than that of theparent polypeptide when assays are performed using practically the sameamount of polypeptides to be compared.

Heterodimerized polypeptides with attenuated, decreased, or loweredbinding to FcγR or FcγR-binding activity refers to those that bind toFcγR with a substantially weaker binding activity than that ofhomodimerized polypeptides when assays are performed using practicallythe same amount of polypeptides to be compared.

For example, in the KD values measured by the above-mentionedmeasurement methods, the KD value ratio (KD value of the parentpolypeptide/KD value of the mutated polypeptide) is preferably 0.99 orless, 0.95 or less, 0.9 or less, 0.8 or less, 0.7 or less, 0.5 or less,0.3 or less, and 0.1 or less, and more preferably, 0.08 or less, 0.05 orless, 0.02 or less, 0.01 or less, and 0.001 or less. Herein, the KDvalue ratio is also called the KD ratio.

The KD values measured by the above-mentioned measurement methods arepreferably increased by 1 μM or more, and more preferably increased by10 μM, 100 μM, 1 nM or more, 2 nM or more, 3 nM or more, 5 nM or more,10 nM or more, 20 nM or more, 50 nM or more, 100 nM or more, or 1 μM ormore. The KD values measured by the above-mentioned measurement methodsare preferably 1 μM or more, and preferably 10 μM or more, 100 μM ormore, 1 nM or more, 10 nM or more, 100 nM or more, 500 nM or more, 1 μMor more, 3 μM or more, or 5 μM or more.

Enhancement, increase, or improvement of binding to FcγR or FcγR-bindingactivity refers to binding to FcγR with a substantially stronger bindingactivity than that of the parent polypeptide when assays are performedusing practically the same amount of polypeptides to be compared.

Heterodimerized polypeptides with enhanced, increased, or improvedbinding to FcγR or FcγR-binding activity refers to those that bind toFcγR with a substantially stronger binding activity than that ofhomodimerized polypeptides when assays are performed using practicallythe same amount of polypeptides to be compared.

For example, in the KD values measured by the above-mentionedmeasurement methods, the KD value ratio (KD value of the parentpolypeptide/KD value of the mutated polypeptide) is preferably 1.1 ormore, 1.2 or more, 1.3 or more, 1.5 or more, 1.8 or more, 2 or more, or3 or more, and more preferably, 5 or more, 10 or more, 100 or more, 250or more, or 1000 or more. Herein, the KD value ratio is also called KDratio.

The KD values measured by the above-mentioned measurement methods arepreferably decreased by 1 μM or more, and more preferably decreased by10 μM, 100 μM, 1 nM or more, 2 nM or more, 3 nM or more, 5 nM or more,10 nM or more, 20 nM or more, 50 nM or more, 100 nM or more, or 1 μM ormore.

The KD values measured by the above-mentioned measurement methods arepreferably 5 μM or less, and more preferably, 3 μM or less, 1 μM orless, 0.5 μM or less, 0.1 μM or less, 0.01 μM or less, 1 nM or less, 0.1nM or less, 0.001 nM or less, or 1 μM or less.

In the present invention, when alteration of Fc region function of thepolypeptide is enhancement of binding activity to an Fcγ receptor, aminoacid mutations may be introduced into the amino acid sequences of thefirst polypeptide and/or the second polypeptide constituting theaforementioned Fc region. The type and range of the amino acid mutationto be introduced are not particularly limited.

The strength of interaction between an antibody Fc region and FcγR hasbeen reported to depend on the Zn²⁺ ion concentration (ImmunologyLetters 143 (2012) 60-69). The antibody shows a stronger interactionbetween the Fc region and FcgR when the Zn²⁺ ion concentration of the Fcregion is higher. Chelation of Zn²⁺ by His310 and His435 present in CH3of the antibody Fc region opens up each CH2 domain of the Fc region at adistal position. This facilitates interaction between the CH2 domain andFcgR, and the interaction between the Fc region and FcgR is enhanced. Anon-limiting embodiment of the antibody of the present inventionincludes an Fc region in which His at position 310, His at position 435,His at position 433, and/or Asn at position 434 (EU numbering) ischelated with Zn²⁺.

When the Fcγ receptor is FcγRIa, at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Region i of Tables 2-1 and 2-2 herein may be introducedinto the amino acid sequences of the first polypeptide and/or the secondpolypeptide constituting the Fc region. Furthermore, when the Fcγreceptor is FcγRIa, at least one or more amino acid mutations selectedfrom the group consisting of the amino acid mutations described inRegion ii of Tables 2-1, 2-2, and 2-3 herein may be introduced into theamino acid sequences of the first polypeptide and/or the secondpolypeptide constituting the Fc region.

When the Fcγ receptor is FcγRIIa R, at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Region i of Tables 3-1 and 3-2 herein may be introducedinto the amino acid sequences of the first polypeptide and/or the secondpolypeptide constituting the Fc region. Furthermore, when the Fcγreceptor is FcγRIIa R, at least one or more amino acid mutationsselected from the group consisting of the amino acid mutations describedin Region ii of Tables 3-1 and 3-2 herein may be introduced into theamino acid sequences of the first polypeptide and/or the secondpolypeptide constituting the Fc region.

When the Fcγ receptor is FcγRIIa H, at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Region i of Table 4 herein may be introduced into the aminoacid sequences of the first polypeptide and/or the second polypeptideconstituting the Fc region. Furthermore, when the Fcγ receptor isFcγRIIa H, at least one or more amino acid mutations selected from thegroup consisting of the amino acid mutations described in Region ii ofTable 4 herein may be introduced into the amino acid sequences of thefirst polypeptide and/or the second polypeptide constituting the Fcregion.

When the Fcγ receptor is FcγRIIb, at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Region i of Table 5 herein may be introduced into the aminoacid sequences of the first polypeptide and/or the second polypeptideconstituting the Fc region. Furthermore, when the Fcγ receptor isFcγRIIb, at least one or more amino acid mutations selected from thegroup consisting of the amino acid mutations described in Region ii ofTable 5 herein may be introduced into the amino acid sequences of thefirst polypeptide and/or the second polypeptide constituting the Fcregion.

When the Fcγ receptor is FcγRIIIa, at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Region i of Table 6 herein may be introduced into the aminoacid sequences of the first polypeptide and/or the second polypeptideconstituting the Fc region. Furthermore, when the Fcγ receptor isFcγRIIIa, at least one or more amino acid mutations selected from thegroup consisting of the amino acid mutations described in Region ii ofTable 6 herein may be introduced into the amino acid sequences of thefirst polypeptide and/or the second polypeptide constituting the Fcregion. Furthermore, when the Fcγ receptor is FcγRIIIa, morespecifically, at least one or more (for example, two or three) aminoacid mutations selected from the group consisting of substitution ofamino acid L at position 234 (EU numbering) with Y; substitution ofamino acid L at position 235 (EU numbering) with Y or Q; substitution ofamino acid G at position 236 (EU numbering) with W; substitution ofamino acid S at position 239 (EU numbering) with M; substitution ofamino acid H at position 268 (EU numbering) with D; substitution ofamino acid D at position 270 (EU numbering) with E; substitution ofamino acid S at position 298 (EU numbering) with A; substitution ofamino acid K at position 326 (EU numbering) with D; substitution ofamino acid A at position 327 (EU numbering) with D; substitution ofamino acid L at position 328 (EU numbering) with W; substitution ofamino acid A at position 330 (EU numbering) with M or K; andsubstitution of amino acid K at position 334 (EU numbering) with E or Lmay be introduced into the amino acid sequences of the first polypeptideand/or the second polypeptide constituting the Fc region. Even morespecifically, when the Fcγ receptor is FcγRIIIa, at least one or more(for example, two or three) amino acid mutations selected from the groupconsisting of substitution of amino acid S at position 239 (EUnumbering) with D; substitution of amino acid A at position 330 (EUnumbering) with L; and substitution of amino acid I at position 332 (EUnumbering) with E may be introduced into the amino acid sequence ofeither one of the polypeptides constituting the Fc region, the firstpolypeptide or the second polypeptide, and at least one or more (forexample, two or three) amino acid mutations selected from the groupconsisting of substitution of amino acid L at position 234 (EUnumbering) with Y; substitution of amino acid G at position 236 (EUnumbering) with W; and substitution of amino acid S at position 298 (EUnumbering) with A may be introduced into the amino acid sequence of theother polypeptide.

Even more specifically, when the Fcγ receptor is FcγRIIIa, a mutationmay be introduced into at least one or more (for example, two or three)amino acids selected from among Leu at position 234, Leu at position235, Gly at position 236, Ser at position 239, His at position 268, Aspat position 270, Ser at position 298, Ala at position 327, Leu atposition 328, and Lys at position 334 (EU numbering) in the amino acidsequence of either one of the polypeptides constituting the Fc region,the first polypeptide or the second polypeptide; and a mutation may beintroduced into at least one or more (for example, two or three) aminoacids selected from among Asp at position 270, Lys at position 326, Alaat position 330, and Lys at position 334 (EU numbering) in the aminoacid sequence of the other polypeptide.

The amino acid to be altered may be selected appropriately, andpreferably at least one or more (for example, two or three) amino acidmutations selected from the group consisting of:

substitution of amino acid L at position 234 (EU numbering) with Y;

substitution of amino acid L at position 235 (EU numbering) with Y or Q;

substitution of amino acid G at position 236 (EU numbering) with W;

substitution of amino acid S at position 239 (EU numbering) with M;

substitution of amino acid H at position 268 (EU numbering) with D;

substitution of amino acid D at position 270 (EU numbering) with E;

substitution of amino acid S at position 298 (EU numbering) with A;

substitution of amino acid A at position 327 (EU numbering) with D;

substitution of amino acid L at position 328 (EU numbering) with W; and

substitution of amino acid K at position 334 (EU numbering) with L maybe introduced into the amino acid sequence of either one of thepolypeptides constituting the Fc region, the first polypeptide or thesecond polypeptide; and at least one or more (for example, two or three)amino acid mutations selected from the group consisting of:substitution of amino acid D at position 270 (EU numbering) with E;substitution of amino acid K at position 326 (EU numbering) with D;substitution of amino acid A at position 330 (EU numbering) with M or K;andsubstitution of amino acid K at position 334 (EU numbering) with E maybe introduced into the amino acid sequence of the other polypeptide.

More preferably, any one set of mutations of (i) to (vi) may beintroduced into the amino acid sequence of either one of thepolypeptides constituting the Fc region, the first polypeptide or thesecond polypeptide; and any one set of mutations of (vii) to (ix) may beintroduced into the amino acid sequence of the other polypeptide:

(i) substitution of amino acid L at position 234 (EU numbering) with Y;

-   -   substitution of amino acid L at position 235 (EU numbering) with        Y;    -   substitution of amino acid G at position 236 (EU numbering) with        W;    -   substitution of amino acid H at position 268 (EU numbering) with        D; and    -   substitution of amino acid S at position 298 (EU numbering) with        A;        (ii) substitution of amino acid L at position 234 (EU numbering)        with Y;    -   substitution of amino acid L at position 235 (EU numbering) with        Y;    -   substitution of amino acid G at position 236 (EU numbering) with        W;    -   substitution of amino acid H at position 268 (EU numbering) with        D;    -   substitution of amino acid D at position 270 (EU numbering) with        E; and    -   substitution of amino acid S at position 298 (EU numbering) with        A;        (iii) substitution of amino acid L at position 234 (EU        numbering) with Y;    -   substitution of amino acid L at position 235 (EU numbering) with        Q;    -   substitution of amino acid G at position 236 (EU numbering) with        W;    -   substitution of amino acid S at position 239 (EU numbering) with        M;    -   substitution of amino acid H at position 268 (EU numbering) with        D;    -   substitution of amino acid D at position 270 (EU numbering) with        E; and    -   substitution of amino acid S at position 298 (EU numbering) with        A;        (iv) substitution of amino acid L at position 234 (EU numbering)        with Y;    -   substitution of amino acid L at position 235 (EU numbering) with        Y;    -   substitution of amino acid G at position 236 (EU numbering) with        W;    -   substitution of amino acid H at position 268 (EU numbering) with        D;    -   substitution of amino acid S at position 298 (EU numbering) with        A; and    -   substitution of amino acid A at position 327 (EU numbering) with        D;        (v) substitution of amino acid L at position 234 (EU numbering)        with Y;    -   substitution of amino acid L at position 235 (EU numbering) with        Y;    -   substitution of amino acid G at position 236 (EU numbering) with        W;    -   substitution of amino acid S at position 239 (EU numbering) with        M;    -   substitution of amino acid H at position 268 (EU numbering) with        D;    -   substitution of amino acid S at position 298 (EU numbering) with        A; and    -   substitution of amino acid A at position 327 (EU numbering) with        D;        (vi) substitution of amino acid L at position 234 (EU numbering)        with Y;    -   substitution of amino acid L at position 235 (EU numbering) with        Y;    -   substitution of amino acid G at position 236 (EU numbering) with        W;    -   substitution of amino acid S at position 239 (EU numbering) with        M;    -   substitution of amino acid H at position 268 (EU numbering) with        D;    -   substitution of amino acid S at position 298 (EU numbering) with        A;    -   substitution of amino acid A at position 327 (EU numbering) with        D;    -   substitution of amino acid L at position 328 (EU numbering) with        W; and    -   substitution of amino acid K at position 334 (EU numbering) with        L;        (vii) substitution of amino acid K at position 326 (EU        numbering) with D;    -   substitution of amino acid A at position 330 (EU numbering) with        M; and    -   substitution of amino acid K at position 334 (EU numbering) with        E;        (viii) substitution of amino acid D at position 270 (EU        numbering) with E;    -   substitution of amino acid K at position 326 (EU numbering) with        D;    -   substitution of amino acid A at position 330 (EU numbering) with        M; and    -   substitution of amino acid K at position 334 (EU numbering) with        E;        (ix) substitution of amino acid D at position 270 (EU numbering)        with E;    -   substitution of amino acid K at position 326 (EU numbering) with        D;    -   substitution of amino acid A at position 330 (EU numbering) with        K; and    -   substitution of amino acid K at position 334 (EU numbering) with        E.

Selectivity of binding activity can be determined by measuring thebinding activities of the polypeptide towards the respective Fcγreceptor isoforms, and then determining their ratios. For example, theamount of binding and KD value towards an FcγR can be used as anindicator of binding activity.

Herein, “improvement of selectivity of binding activity” means that, forexample, the ratio of binding activities of a test polypeptide to Fcγreceptor isoforms (binding activity of the test polypeptide to a firstFcγ receptor isoform/binding activity of the test polypeptide to thesecond Fcγ receptor isoform) is increased by 0.1 or more, or preferably0.2 or more, 0.5 or more, 1 or more, 2 or more, 3 or more, 5 or more, 7or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 30 ormore, 50 or more, 70 or more, 100 or more, 150 or more, 200 or more, 500or more, or 1000 or more when compared to the ratio of bindingactivities of a parent polypeptide of the test polypeptide to the Fcγreceptor isoforms (binding activity of the parent polypeptide of thetest polypeptide to the first Fcγ receptor isoform/binding activity ofthe parent polypeptide of the test polypeptide to the second Fcγreceptor isoform) determined based on the above-mentioned measurementmethod. Furthermore, decreased Fcγ receptor isoform selectivity meansthat, for example, the ratio of binding activities of a test polypeptideto Fcγ receptor isoforms is reduced by 0.1 or more, or preferably 0.2 ormore, 0.5 or more, 1 or more, 2 or more, 3 or more, 5 or more, 7 ormore, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 30 ormore, 50 or more, 70 or more, 100 or more, 150 or more, 200 or more, 500or more, or 1000 or more when compared to the ratio of bindingactivities of the parent polypeptide of the test polypeptide to the Fcγreceptor isoforms determined based on the above-mentioned measurementmethod.

Herein, as an indicator of selectivity, for example, the A/I ratio whichshows the ratio of binding activities towards activating FcγR andinhibitory FcγR can also be used. The values obtained by dividing the KDof the test polypeptide for FcγRIIb by the KD of the test polypeptidefor FcγRIIa H type or R type were used as the respective A/I ratios. TheA/I ratio is preferably 1.1 or more, 1.5 or more, 2 or more, 3 or more,or 5 or more, and more preferably 6 or more, 8 or more, or 9 or more.

Herein, as an indicator of selectivity, for example, the FcγRIIIaF/FcγRIIb ratio which is a value obtained by dividing the KD for FcγRIIbby the KD for FcγRIIIa F can be used. The values obtained by dividingthe KD of the test polypeptide for FcγRIIb by the KD of the testpolypeptide for FcγRIIIa were defined as the respective FcγRIIIaF/FcγRIIb ratios. The FcγRIIIa F/FcγRIIb ratio is preferably 1.1 ormore, 1.5 or more, 2 or more, 3 or more, or 5 or more, and morepreferably 10 or more, 20 or more, 30 or more, 40 or more, 50 or more,60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 ormore, 120 or more, 130 or more, 140 or more, 150 or more, 200 or more,210 or more, 220 or more, 230 or more, or 240 or more.

In the present invention, when the alteration of Fc region function ofthe polypeptide is the improvement of selectivity of binding activity toan Fey receptor, an amino acid mutation may be introduced into the aminoacid sequence of the first polypeptide and/or the second polypeptideconstituting the Fc region. The type and range of the amino acidmutation to be introduced is not particularly limited.

In a case where the activating Fey receptor is FcγRIa, the inhibitoryFey receptor is FcγRIIb, and the improvement of selectivity is selectiveenhancement of binding activity to FcγRIa than to FcγRIIb, at least oneor more amino acid mutations selected from the group consisting of theamino acid mutations described in Region a of Tables 19-1, 19-2, 19-3,and 19-4 herein may be introduced into the amino acid sequences of thefirst polypeptide and/or the second polypeptide constituting the Fcregion. Furthermore, when the improvement of selectivity is selectiveenhancement of binding activity to FcγRIa than to FcγRIIb, at least oneor more amino acid mutations selected from the group consisting of theamino acid mutations described in Region b of Tables 19-1, 19-2, 19-3,19-4, and 19-5 herein may be introduced into the amino acid sequences ofthe first polypeptide and/or the second polypeptide constituting the Fcregion.

In a case where the activating Fey receptor is FcγRIa, the inhibitoryFey receptor is FcγRIIb, and the improvement of selectivity is selectivereduction of binding activity to FcγRIa than to FcγRIIb, at least one ormore amino acid mutations selected from the group consisting of theamino acid mutations described in Region c of Tables 23-1 and 23-2herein may be introduced into the amino acid sequences of the firstpolypeptide and/or the second polypeptide constituting the Fc region.Furthermore, when the improvement of selectivity is selective reductionof binding activity to FcγRIa than to FcγRIIb, at least one or moreamino acid mutations selected from the group consisting of the aminoacid mutations described in Region d of Tables 23-1 and 23-2 herein maybe introduced into the amino acid sequences of the first polypeptideand/or the second polypeptide constituting the Fc region.

In a case where the activating Fey receptor is FcγRIIa R, the inhibitoryFey receptor is FcγRIIb, and the improvement of selectivity is selectiveenhancement of binding activity to FcγRIIa R than to FcγRIIb, at leastone or more amino acid mutations selected from the group consisting ofthe amino acid mutations described in Region a of Table 20-1 herein maybe introduced into the amino acid sequences of the first polypeptideand/or the second polypeptide constituting the Fc region. Furthermore,when the improvement of selectivity is selective enhancement of bindingactivity to FcγRIIa R than to FcγRIIb, at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Region b of Tables 20-1, 20-2, and 20-3 herein may beintroduced into the amino acid sequences of the first polypeptide and/orthe second polypeptide constituting the Fc region.

In a case where the activating Fcγ receptor is FcγRIIa R, the inhibitoryFcγ receptor is FcγRIIb, and the improvement of selectivity is selectivereduction of binding activity to FcγRIIa R than to FcγRIIb, at least oneamino acid mutations selected from the group consisting of the aminoacid mutations described in Region c of Table 24-1 herein may beintroduced into the amino acid sequences of the first polypeptide and/orthe second polypeptide constituting the Fc region. Furthermore, when theimprovement of selectivity is selective reduction of binding activity toFcγRIIa R than to FcγRIIb, at least one or more amino acid mutationsselected from the group consisting of the amino acid mutations describedin Region d of Tables 24-1 and 24-2 herein may be introduced into theamino acid sequences of the first polypeptide and/or the secondpolypeptide constituting the Fc region.

In a case where the activating Fcγ receptor is FcγRIIa H, the inhibitoryFcγ receptor is FcγRIIb, and the improvement of selectivity is selectiveenhancement of binding activity to FcγRIIa H than to FcγRIIb, at leastone or more amino acid mutations selected from the group consisting ofthe amino acid mutations described in Region a of Table 21-1 herein maybe introduced into the amino acid sequences of the first polypeptideand/or the second polypeptide constituting the Fc region. Furthermore,when the improvement of selectivity is selective enhancement of bindingactivity to FcγRIIa H than to FcγRIIb, at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Region b of Tables 21-1, 21-2, and 21-3 herein may beintroduced into the amino acid sequences of the first polypeptide and/orthe second polypeptide constituting the Fc region.

In a case where the activating Fcγ receptor is FcγRIIa H, the inhibitoryFcγ receptor is FcγRIIb, and the improvement of selectivity is selectivereduction of binding activity to FcγRIIa H than to FcγRIIb, at least oneor more amino acid mutations selected from the group consisting of theamino acid mutations described in Region c of Tables 25-1 and 25-2herein may be introduced into the amino acid sequences of the firstpolypeptide and/or the second polypeptide constituting the Fc region.Furthermore, when the improvement of selectivity is selective reductionof binding activity to FcγRIIa H than to FcγRIIb, at least one or moreamino acid mutations selected from the group consisting of the aminoacid mutations described in Region d of Tables 25-1, 25-2, and 25-3herein may be introduced into the amino acid sequences of the firstpolypeptide and/or the second polypeptide constituting the Fc region.

In a case where the activating Fcγ receptor is FcγRIIIa, the inhibitoryFcγ receptor is FcγRIIb, and the improvement of selectivity is selectiveenhancement of binding activity to FcγRIIIa than to FcγRIIb, at leastone or more amino acid mutations selected from the group consisting ofthe amino acid mutations described in Region a of Table 22-1 herein maybe introduced into the amino acid sequences of the first polypeptideand/or the second polypeptide constituting the Fc region. Furthermore,when the improvement of selectivity is selective enhancement of bindingactivity to FcγRIIIa than to FcγRIIb, at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Region b of Tables 22-1, 22-2, and 22-3 herein may beintroduced into the amino acid sequences of the first polypeptide and/orthe second polypeptide constituting the Fc region.

In a case where the activating Fcγ receptor is FcγRIIIa, the inhibitoryFcγ receptor is FcγRIIb, and the improvement of selectivity is selectivereduction of binding activity to FcγRIIIa than to FcγRIIb, at least oneor more amino acid mutations selected from the group consisting of theamino acid mutations described in Region c of Tables 26-1 and 26-2herein may be introduced into the amino acid sequences of the firstpolypeptide and/or the second polypeptide constituting the Fc region.Furthermore, when the improvement of selectivity is selective reductionof binding activity to FcγRIIIa than to FcγRIIb, at least one or moreamino acid mutations selected from the group consisting of the aminoacid mutations described in Region d of Tables 26-1, 26-2, 26-3, and26-4 herein may be introduced into the amino acid sequences of the firstpolypeptide and/or the second polypeptide constituting the Fc region.

Herein, the selective enhancement of binding activity to a desired Fcγreceptor means any of the following cases:

-   (i) binding activity to a desired Fcγ receptor is enhanced, and    binding activities to receptors other than the desired Fcγ receptor    are unchanged or decreased;-   (ii) binding activity to a desired Fcγ receptor is enhanced, and    binding activities to receptors other than the desired Fcγ receptor    are also enhanced, but the degree of enhancement of binding activity    to receptors other than the desired Fcγ receptor is less than the    degree of enhancement of binding activity to the desired Fcγ    receptor; or-   (iii) binding activity to a desired Fcγ receptor is decreased, but    the degree of reduction of binding activity is less than the degree    of reduction of binding activities to Fcγ receptors other than the    desired Fcγ receptor.

Furthermore, selective reduction of binding activity to a desired Fcγreceptor means any of the following cases:

-   (i) binding activity to a desired Fcγ receptor is decreased, and    binding activities to receptors other than the desired Fcγ receptor    are unchanged or enhanced;-   (ii) binding activity to a desired Fcγ receptor is decreased, and    binding activities to receptors other than the desired Fcγ receptor    are also decreased, but the degree of reduction of binding activity    to receptors other than the desired Fcγ receptor is less than the    degree of reduction of binding activity to the desired Fcγ receptor;    or-   (iii) binding activity to a desired Fcγ receptor is enhanced, but    the degree of enhancement of binding activity is less than the    degree of enhancement of binding activities to Fcγ receptors other    than the desired Fcγ receptor.

Herein, physicochemical stability of a polypeptide means, for example,thermodynamic stability of a polypeptide, which can be determined using,for example, the Tm value of the CH2 domain as an indicator. Tm valuescan be measured by circular dichroism (CD), differential scanningcalorimeter (DSC), or differential scanning fluorimetry (DSF).

The change in mean residue molar ellipticity (θ) that accompanies a risein temperature is measured by CD to calculate the Tm value. Themeasuring instrument includes, for example, a circular dichroismdispersion meter (JASCO Corporation). When CD spectra are measured at asuitable wavelength (for example, 208 nm or 222 nm) while increasing thetemperature, θ increases at a certain temperature, and becomes aconstant value at higher temperatures. The temperature at which amidpoint value between low-temperature θ and high-temperature θ is takenas Tm. For measurement, it is possible to use, for example, a proteinsolution prepared using citric acid, Tris, phosphate solution, or such,at a concentration of several hundred ug/mL.

DSC measures the change in calorie that accompanies a rise intemperature to calculate the Tm value. The measuring instrument includesMicroCal VP-DSC and Micro Cal Capillary DSC (both from DKSH Japan). Aprotein solution and a buffer are filled in measurement cells, and whentemperature differences among the cells are measured while raising thetemperature, a change to endothermic reaction is observed starting at acertain temperature. This temperature is taken to be Tm. Formeasurement, it is possible to use, for example, a protein solutionprepared using citrate buffer, TBS, PBS, histidine buffer, or such at aconcentration of several ten ug/mL to several hundred ug/mL.

DSF detects exposure of hydrophobic residues that accompanies a rise intemperature by using a fluorescent reagent (for example, SYPRO Orange)that specifically binds to hydrophobic residues to calculate the Tmvalue. A protein solution and a fluorescence reagent are mixed atappropriate ratios, and when fluorescence intensities are measured whileraising the temperature using an RT-PCR instrument, increase influorescence intensity is observed at a certain temperature. Thistemperature is taken to be Tm. Examples of the measuring instrumentinclude Rotor-Gene Q (QIAGEN), and CFX96 real-time PCR analysis system(Bio-Rad). For measurement, it is possible to use, for example, aprotein solution prepared using PBS, histidine buffer, or such at aconcentration of several ten ug/mL to several hundred ug/mL.

Herein, improved physicochemical stability of a polypeptide means that,for example, the Tm value of the CH2 domain in the Fc region of a testpolypeptide determined based on the above-mentioned measurement methodis increased by 0.1 degrees or more, preferably 0.2 degrees or more, 0.3degrees or more, 0.4 degrees or more, 0.5 degrees or more, 1 degree ormore, 2 degrees or more, 3 degrees or more, 4 degrees or more, 5 degreesor more, or 10 degrees or more compared to the Tm value of the CH2domain in the Fc region of a control polypeptide. Furthermore, improvedphysical stability of a polypeptide refers to suppressed reduction ofphysical stability of a polypeptide; and for example, reduction of theTm value of the CH2 domain in the Fc region of a test polypeptide issuppressed relative to the Tm value of the CH2 domain in the Fc regionof a control polypeptide by 0.1 degrees or more, preferably 0.2 degreesor more, 0.3 degrees or more, 0.4 degrees or more, 0.5 degrees or more,1 degree or more, 2 degrees or more, 3 degrees or more, 4 degrees ormore, 5 degrees or more, or 10 degrees or more, as determined based onthe above-mentioned measurement method.

Herein, “reduction of physical stability of a polypeptide” means thatthe Tm value of the CH2 domain in the Fc region of a test polypeptidedetermined based on the above-mentioned measurement method is decreasedby 0.1 degrees or more, preferably 0.2 degrees or more, 0.3 degrees ormore, 0.4 degrees or more, 0.5 degrees or more, 1 degree or more, 2degrees or more, 3 degrees or more, 4 degrees or more, 5 degrees ormore, or 10 degrees or more compared to the Tm value of CH2 domain inthe Fc region of a control peptide.

The present invention also comprises a polypeptide comprising an Fcregion, wherein the polypeptide is characterized in that the Fc regionis composed of a heterodimer comprising a first polypeptide and a secondpolypeptide, and wherein a function of the Fc region is altered comparedto that of a polypeptide characterized in that the Fc region is composedof a homodimer comprising only the first polypeptide or when compared tothat of a polypeptide characterized in that the Fc region is composed ofa homodimer comprising only the second polypeptide.

In the polypeptide, the alteration of Fc region function may be analteration that further improves physicochemical stability, in additionto at least one or more alterations selected from the group consistingof enhancement of binding activity, reduction of binding, andimprovement of selectivity of binding activity of the polypeptide to anFcγ receptor; and as long as any of these functions is altered, it canbe said that the Fc region function of the present invention is altered.

In the present invention, the phrase “when amino acid mutations areintroduced into the Fc region in both the first polypeptide and thesecond polypeptide, the Fc region function is not altered” means thatwhen the same amino acid mutations are introduced into both the firstpolypeptide and the second polypeptide, the desired function is notimproved. For example, it means that when one intends to enhance thebinding activity of a polypeptide to an Fcγ receptor, the bindingactivity does not change or is decreased; when one intends to reduce thebinding activity, the binding activity does not change or is enhanced;when one intends to improve selectivity of the binding activity, theselectivity is not improved; and when one intends to improvephysicochemical stability of the polypeptide, the stability does notchange or is decreased. Regarding the amino acid mutation, the phrase“when it is introduced into only one of the Fc regions, the Fc regionfunction is altered” means that the desired function is improved whenthe amino acid mutation is introduced only into either the firstpolypeptide or the second polypeptide. For example, it means that whenone intends to enhance the binding activity of a polypeptide to an Fcγreceptor, the binding activity is enhanced; when one intends to reducethe binding activity, the binding activity is decreased; when oneintends to improve selectivity of binding activity, the selectivity isimproved; and when one intends to improve physicochemical stability of apolypeptide, the stability is improved.

The present invention also includes a polypeptide which is characterizedin that the Fc region is composed of a heterodimer comprising a firstpolypeptide and a second polypeptide, and which is characterized inhaving a higher Tm than that of a polypeptide characterized in that theFc region is composed of a homodimer comprising only the firstpolypeptide or that of a polypeptide characterized in that the Fc regionis composed of a homodimer comprising only the second polypeptide. Alongwith an alteration that improves physicochemical stability, i.e., havinga high Tm, the polypeptide may also have additional alterations to theFc region function.

In a case where the additional alteration of Fc region function isenhancement of FcγRIa-binding activity, at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Tables 31-1, 31-2, and 31-3 herein may be introduced intothe amino acid sequences of the first polypeptide and/or the secondpolypeptide constituting the Fc region.

In a case where the additional alteration of Fc region function isenhancement of FcγRIIa R-binding activity, at least one or more aminoacid mutations selected from the group consisting of the amino acidmutations described in Tables 32-1 and 32-2 herein may be introducedinto the amino acid sequences of the first polypeptide and/or the secondpolypeptide constituting the Fc region.

In a case where the additional alteration of Fc region function isenhancement of FcγRIIa H-binding activity, at least one or more aminoacid mutations selected from the group consisting of the amino acidmutations described in Tables 33-1 and 33-2 herein may be introducedinto the amino acid sequences of the first polypeptide and/or the secondpolypeptide constituting the Fc region.

In a case where the additional alteration of Fc region function isenhancement of FcγRIIb-binding activity, at least one or more amino acidmutations selected from the group consisting of the amino acid mutationsdescribed in Tables 34-1 and 34-2 herein may be introduced into theamino acid sequences of the first polypeptide and/or the secondpolypeptide constituting the Fc region.

In a case where the additional alteration of Fc region function isenhancement of FcγRIIIa-binding activity, at least one or more aminoacid mutations selected from the group consisting of the amino acidmutations described in Tables 35-1 and 35-2 herein may be introducedinto the amino acid sequences of the first polypeptide and/or the secondpolypeptide constituting the Fc region.

In the present invention, the combination of the first polypeptide andthe second polypeptide into which amino acid mutations are introduced isnot particularly limited, and examples include combinations of differenttypes/or the same type of polypeptides selected from the polypeptidesdescribed in SEQ ID NOs: 2 to 4 and 6 to 60. In addition, the preferredexamples include a combination of polypeptides comprising the firstpolypeptide and the second polypeptide described in the Examples herein(a combination of the H chains of two antibodies and the L chain of asingle antibody).

The polypeptide of the present invention may be an antigen-bindingmolecule. In the present invention, while the antibody-binding moleculeis not particularly limited in type, preferred examples include anantibody, a bispecific antibody, or an Fc fusion molecule such as apeptide Fc fusion protein or a scaffold Fc fusion protein.

<Antibody>

Furthermore, an antibody is provided as a polypeptide of the presentinvention.

The term “antibody/antibodies” in the present invention is used in thebroadest sense, and as long as the desired biological activity is shown,it comprises any antibody such as monoclonal antibodies (includingfull-length monoclonal antibodies), polyclonal antibodies, antibodyvariants, antibody fragments, polyspecific antibodies (for example,bispecific antibodies), chimeric antibodies, and humanized antibodies.

Regarding the antibodies of the present invention, the antigen type andantibody origin are not limited, and they may be any type of antibodies.The origin of the antibodies is not particularly limited, but examplesinclude human antibodies, mouse antibodies, rat antibodies, and rabbitantibodies.

Methods for producing the antibodies are well known to those skilled inthe art, and for example, monoclonal antibodies may be produced by thehybridoma method (Kohler and Milstein, Nature 256: 495 (1975)), or therecombination method (U.S. Pat. No. 4,816,567). Alternatively, they maybe isolated from a phage antibody library (Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1991)).Alternatively, they may be isolated from a single B cell clone (N.Biotechnol. 28(5): 253-457 (2011)).

A humanized antibody is also called a reshaped human antibody.Specifically, humanized antibodies prepared by grafting the CDRs of anon-human animal antibody such as a mouse antibody to a human antibodyand such are known. Common genetic engineering technologies forobtaining humanized antibodies are also known. Specifically, forexample, overlap extension PCR is known as a method for grafting mouseantibody CDRs to human FRs.

A vector for expressing a humanized antibody can be produced byinserting a DNA encoding an antibody variable region in which three CDRsand four FRs are ligated and a DNA encoding a human antibody constantregion into an expression vector so that these DNAs are fused in frame.After this integration vector is transfected into a host to establishrecombinant cells, these cells are cultured, and the DNA encoding thehumanized antibody is expressed to produce the humanized antibody in theculture of the cells (see, European Patent Publication No. EP 239,400,and International Patent Publication No. WO 1996/002576).

As necessary, an amino acid residue in an FR may be substituted so thatthe CDRs of a reshaped human antibody form an appropriateantigen-binding site. For example, a mutation can be introduced into theamino acid sequence of an FR by applying the PCR method used forgrafting mouse CDRs to human FRs.

A desired human antibody can be obtained by DNA immunization using atransgenic animal having the complete repertoire of human antibody genes(see International Publication Nos. WO 1993/012227, WO 1992/003918, WO1994/002602, WO 1994/025585, WO 1996/034096, and WO 1996/033735) as ananimal for immunization.

Furthermore, technologies for obtaining a human antibody by panningusing a human antibody library are known. For example, a human antibodyV region is expressed on the surface of a phage as a single-chainantibody (scFv) by the phage display method. The scFv-expressing phagethat binds to the antigen can be selected. The DNA sequence that encodesthe V region of the antigen-bound human antibody can be determined byanalyzing the genes of the selected phage. After determining the DNAsequence of the scFv that binds to the antigen, an expression vector canbe prepared by fusing the V-region sequence in-frame with the sequenceof a desired human antibody C region, and then inserting this into asuitable expression vector. The expression vector is introduced intosuitable expression cells such as those described above, and the humanantibody can be obtained by expressing the human antibody-encoding gene.These methods are already known (see, International Publication Nos. WO1992/001047, WO 1992/020791, WO 1993/006213, WO 1993/011236, WO1993/019172, WO 1995/001438, and WO 1995/15388).

Variable regions constituting the antibodies of the present inventioncan be variable regions that recognize any antigen.

Herein, there is no particular limitation on the antigen, and it may beany antigens. Examples of the antigen include 17-IA, 4-1 BB, 4Dc,6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 Adenosine Receptor, A33, ACE,ACE-2, Activin, Activin A, Activin AB, Activin B, Activin C, ActivinRIA, Activin RIA ALK-2, Activin RIB ALK-4, Activin RITA, Activin RIIB,ADAM, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAMS, ADAMS, ADAMTS,ADAMTS4, ADAMTS5, Addressins, adiponectin, ADP ribosyl cyclase-1, aFGF,AGE, ALCAM, ALK, ALK-1, ALK-7, allergen, alpha1-antichemotrypsin,alpha1-antitrypsin, alpha-synuclein, alpha-V/beta-1 antagonist, aminin,amylin, amyloid beta, amyloid immunoglobulin heavy chain variableregion. amyloid immunoglobulin light chain variable region, Androgen,ANG, angiotensinogen, Angiopoietin ligand-2, antithrombinIII, Anthrax,APAF-1, APE, APJ, apo A1, apo serum amyloid A, Apo-SAA, APP, APRIL, AR,ARC, ART, Artemin, ASPARTIC, Atrial natriuretic factor, Atrialnatriuretic peptide, atrial natriuretic peptides A, atrial natriureticpeptides B, atrial natriuretic peptides C, av/b3 integrin, Ax1, B7-1,B7-2, B7-H, BACE, BACE-1, Bacillus anthracis protective antigen, Bad,BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, BcI, BCMA, BDNF, b-ECGF,beta-2-microglobulin, betalactamase, bFGF, BID, Bik, BIM, BLC, BL-CAM,BLK, B-lymphocyte Stimulator (BIyS), BMP, BMP-2 (BMP-2a), BMP-3(Osteogenin), BMP-4 (BMP-2b), BMP-5, BMP-6 (Vgr-1), BMP-7 (OP-1), BMP-8(BMP-8a), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BMPR-II (BRK-3), BMPs,BOK, Bombesin, Bone-derived neurotrophic factor, bovine growth hormone,BPDE, BPDE-DNA, BRK-2, BTC, B-lymphocyte cell adhesion molecule, C10,C1-inhibitor, C1q, C3, C3a, C4, C5, C5a (complement 5a), CA125, CAD-8,Cadherin-3, Calcitonin, cAMP, Carbonic anhydrase-IX, carcinoembryonicantigen (CEA), carcinoma-associated antigen, Cardiotrophin-1, CathepsinA, Cathepsin B, Cathepsin C/DPPI, Cathepsin D, Cathepsin E, Cathepsin H,Cathepsin L, Cathepsin O, Cathepsin S, Cathepsin V, Cathepsin X/Z/P,CBL, CCI, CCK2, CCL, CCL1/1-309, CCL11/Eotaxin, CCL12/MCP-5,CCL13/MCP-4, CCL14/HCC-1, CCL15/HCC-2, CCL16/HCC-4, CCL17/TARC,CCL18/PARC, CCL19/ELC, CCL2/MCP-1, CCL20/MIP-3-alpha, CCL21/SLC,CCL22/MDC, CCL23/MPIF-1, CCL24/Eotaxin-2, CCL25/TECK, CCL26/Eotaxin-3,CCL27/CTACK, CCL28/MEC, CCL3/M1P-1-alpha, CCL3L1/LD-78-beta,CCL4/MIP-1-beta, CCL5/RANTES, CCL6/C10, CCL7/MCP-3, CCL8/MCP-2,CCL9/10/MTP-1-gamma, CCR, CCR1, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6,CCR7, CCR8, CCR9, CD1, CD10, CD105, CD11a, CD11b, CD11c, CD123, CD13,CD137, CD138, CD14, CD140a, CD146, CD147, CD148, CD15, CD152, CD16,CD164, CD18, CD19, CD2, CD20, CD21, CD22, CD23, CD25, CD26, CD27L, CD28,CD29, CD3, CD30, CD30L, CD32, CD33 (p67 proteins), CD34, CD37, CD38,CD3E, CD4, CD40, CD40L, CD44, CD45, CD46, CD49a, CD49b, CD5, CD51, CD52,CD54, CD55, CD56, CD6, CD61, CD64, CD66e, CD7, CD70, CD74, CD8, CD80(B7-1), CD89, CD95, CD105, CD158a, CEA, CEACAM5, CFTR, cGMP, CGRPreceptor, CINC, CKb8-1, Claudin18, CLC, Clostridium botulinum toxin,Clostridium difficile toxin, Clostridium perfringens toxin, c-Met, CMV,CMV UL, CNTF, CNTN-1, complement factor 3 (C3), complement factor D,corticosteroid-binding globulin, Colony stimulating factor-1 receptor,COX, C-Ret, CRG-2, CRTH2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1/Fractalkine,CX3CR1, CXCL, CXCL1/Gro-alpha, CXCL10, CXCL11/1-TAC,CXCL12/SDF-1-alpha/beta, CXCL13/BCA-1, CXCL14/BRAK, CXCL15/Lungkine.CXCL16, CXCL16, CXCL2/Gro-beta CXCL3/Gro-gamma, CXCL3, CXCL4/PF4,CXCL5/ENA-78, CXCL6/GCP-2, CXCL7/NAP-2, CXCL8/IL-8, CXCL9/Mig,CXCL10/IP-10, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cystatinC, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, Decayaccelerating factor, Delta-like protein ligand 4, des(1-3)-IGF-1 (brainIGF-1), Dhh, DHICA oxidase, Dickkopf-1, digoxin, Dipeptidyl peptidaseIV, DK1, DNAM-1, Dnase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2,EDAR, EGF, EGFR (ErbB-1), EGF like domain containing protein 7,Elastase, elastin, EMA, EMMPRIN, ENA, ENA-78, Endosialin, endothelinreceptor, endotoxin, Enkephalinase, eNOS, Eot, Eotaxin, Eotaxin-2,eotaxini, EpCAM, Ephrin B2/EphB4, Epha2 tyrosine kinase receptor,epidermal growth factor receptor (EGFR), ErbB2 receptor, ErbB3 tyrosinekinase receptor, ERCC, EREG, erythropoietin (EPO), Erythropoietinreceptor, E-selectin, ET-1, Exodus-2, F protein of RSV, F10, F11, F12,F13, F5, F9, Factor Ia, Factor IX, Factor Xa, Factor VII, factor VIII,Factor VIIIc, Fas, FcalphaR, FcepsilonRI, FcgammaIIb, FcgammaRI,FcgammaRIIa, FcgammaRIIIa, FcgammaRIIIb, FcRn, FEN-1, Ferritin, FGF,FGF-19, FGF-2, FGF-2 receptor, FGF-3, FGF-8, FGF-acidic, FGF-basic,FGFR, FGFR-3, Fibrin, fibroblast activation protein (FAP), fibroblastgrowth factor, fibroblast growth factor-10, fibronectin, FL, FLIP,Flt-3, FLT3 ligand, Folate receptor, follicle stimulating hormone (FSH),Fractalkine (CX3C), free heavy chain, free light chain, FZD1, FZD10,FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, G250, Gas 6, GCP-2,GCSF, G-CSF, G-CSF receptor, GD2, GD3, GDF, GDF-1, GDF-15 (MIC-1), GDF-3(Vgr-2), GDF-5 (BMP-14/CDMP-1), GDF-6 (BMP-13/CDMP-2), GDF-7(BMP-12/CDMP-3), GDF-8 (Myostatin), GDF-9, GDNF, Gelsolin, GFAP, GF-CSF,GFR-alpha1, GFR-alpha2, GFR-alpha3, GF-β1, gH envelope glycoprotein,GITR, Glucagon, Glucagon receptor, Glucagon-like peptide 1 receptor,Glut 4, Glutamate carboxypeptidase II, glycoprotein hormone receptors,glycoprotein IIb/IIIa (GP IIb/IIIa), Glypican-3, GM-CSF, GM-CSFreceptor, gp130, gp140, gp72, granulocyte-CSF (G-CSF), GRO/MGSA, Growthhormone releasing factor, GRO-β, GRO-γ, H. pylori, Hapten (NP-cap orNIP-cap), HB-EGF, HCC, HCC 1, HCMV gB envelope glycoprotein, HCMV UL,Hemopoietic growth factor (HGF), Hep B gp120, heparanase, heparincofactor II, hepatic growth factor, Bacillus anthracis protectiveantigen, Hepatitis C virus E2 glycoprotein, Hepatitis E, Hepcidin, Her1,Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus(HSV) gB glycoprotein, HGF, HGFA, High molecular weightmelanoma-associated antigen (HMW-MAA), HIV envelope proteins such asGP120, HIV MIB gp120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HMGB-1,HRG, Hrk, HSP47, Hsp90, HSV gD glycoprotein, human cardiac myosin, humancytomegalovirus (HCMV), human growth hormone (hGH), human serum albumin,human tissue-type plasminogen activator (t-PA), Huntingtin, HVEM, IAP,ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFN-alpha, IFN-beta, IFN-gamma, IgA,IgA receptor, IgE, IGF, IGF binding proteins, IGF-1, IGF-1 R, IGF-2,IGFBP, IGFR, IL, IL-1, IL-10, IL-10 receptors, IL-11, IL-11 receptors,IL-12, IL-12 receptors, IL-13, IL-13 receptors, IL-15, IL-15 receptors,IL-16, IL-16 receptors, IL-17, IL-17 receptors, IL-18 (IGIF), IL-18receptors, IL-1alpha, IL-1beta, IL-1 receptors, IL-2, IL-2 receptors,IL-20, IL-20 receptors, IL-21, IL-21 receptors, IL-23, IL-23 receptors,IL-2 receptors, IL-3, IL-3 receptors, IL-31, IL-31 receptors, IL-3receptors, IL-4, IL-4 receptors IL-5, IL-5 receptors, IL-6, IL-6receptors, IL-7, IL-7 receptors, IL-8, IL-8 receptors, IL-9, IL-9receptors, immunoglobulin immune complex, immunoglobulins, INF-alpha,INF-alpha receptors, INF-beta, INF-beta receptors, INF-gamma, INF-gammareceptors, IFN type-I, IFN type-I receptor, influenza, inhibin, Inhibinα, Inhibin β, iNOS, insulin, Insulin A-chain, Insulin B-chain,Insulin-like growth factor 1, insulin-like growth factor 2, insulin-likegrowth factor binding proteins, integrin, integrin alpha2, integrinalpha3, integrin alpha4, integrin alpha4/beta1, integrin alpha-V/beta-3,integrin alpha-V/beta-6, integrin alpha4/beta7, integrin alpha5/beta1,integrin alpha5/beta3, integrin alpha5/beta6, integrin alphaσ (alphaV),integrin alphaθ, integrin beta1, integrin beta2, integrin beta3(GPIIb-IIIa), IP-10, I-TAC, JE, kalliklein, Kallikrein 11, Kallikrein12, Kallikrein 14, Kallikrein 15, Kallikrein 2, Kallikrein 5, Kallikrein6, Kallikrein L1, Kallikrein L2, Kallikrein L3, Kallikrein L4,kallistatin, KC, KDR, Keratinocyte Growth Factor (KGF), KeratinocyteGrowth Factor-2 (KGF-2), KGF, killer immunoglobulin-like receptor, kitligand (KL), Kit tyrosine kinase, laminin 5, LAMP, LAPP (Amylin,islet-amyloid polypeptide), LAP (TGF-1), latency associated peptide,Latent TGF-1, Latent TGF-1 bpl, LBP, LDGF, LDL, LDL receptor, LECT2,Lefty, Leptin, leutinizing hormone (LH), Lewis-Y antigen, Lewis-Yrelated antigen, LFA-1, LFA-3, LFA-3 receptors, Lfo, LIF, LIGHT,lipoproteins, LIX, LKN, Lptn, L-Selectin, LT-a, LT-b, LTB4, LTBP-1, Lungsurfactant, Luteinizing hormone, Lymphotactin, Lymphotoxin BetaReceptor, Lysosphingolipid receptor, Mac-1, macrophage-CSF (M-CSF),MAdCAM, MAG, MAP2, MARC, maspin, MCAM, MCK-2, MCP, MCP-1, MCP-2, MCP-3,MCP-4, MCP-I (MCAF), M-CSF, MDC, MDC (67 a.a.), MDC (69 a.a.), megsin,Mer, MET tyrosine kinase receptor family, METALLOPROTEASES, Membraneglycoprotein OX2, Mesothelin, MGDF receptor, MGMT, MHC(HLA-DR),microbial protein, MIF, MIG, MIP, MIP-1α, MIP-1β, MIP-3α, MIP-3β, MIP-4,MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15,MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, monocyte attractant protein,monocyte colony inhibitory factor, mouse gonadotropin-associatedpeptide, MPIF, Mpo, MSK, MSP, MUC-16, MUC18, mucin (Mud),Muellerian-inhibiting substance, Mug, MuSK, Myelin associatedglycoprotein, myeloid progenitor inhibitor factor-1 (MPIF-I), NAIP,Nanobody, NAP, NAP-2, NCA 90, NCAD, N-Cadherin, NCAM, Neprilysin, Neuralcell adhesion molecule, neroserpin, Neuronal growth factor (NGF),Neurotrophin-3, Neurotrophin-4, Neurotrophin-6, Neuropilin 1, Neurturin,NGF-beta, NGFR, NKG20, N-methionyl human growth hormone, nNOS, NO,Nogo-A, Nogo receptor, non-structural protein type 3 (NS3) from thehepatitis C virus, NOS, Npn, NRG-3, NT, NT-3, NT-4, NTN, OB, OGG1,Oncostatin M, OP-2, OPG, OPN, OSM, OSM receptors, osteoinductivefactors, osteopontin, OX40L, OX40R, oxidized LDL, p150, p95, PADPr,parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-Cadherin, PCNA,PCSK9, PDGF, PDGF receptor, PDGF-AA, PDGF-AB, PDGF-BB, PDGF-D, PDK-1,PECAM, PEDF, PEM, PF-4, PGE, PGF, PGI2, PGD2, P1GF, PIN, PLA2, Placentagrowth factor, placental alkaline phosphatase (PLAP), placentallactogen, plasminogen activator inhibitor-1, platelet-growth factor,plgR, PLP, poly glycol chains of different size (e.g. PEG-20, PEG-30,PEG40), PP14, prekallikrein, prion protein, procalcitonin, Programmedcell death protein 1, proinsulin, prolactin, Proprotein convertase PC9,prorelaxin, prostate specific membrane antigen (PSMA), Protein A,Protein C, Protein D, Protein S, Protein Z, PS, PSA, PSCA, PsmAr, PTEN,PTHrp, Ptk, PTN, P-selectin glycoprotein ligand-1, R51, RAGE, RANK,RANKL, RANTES, relaxin, Relaxin A-chain, Relaxin B-chain, renin,respiratory syncytial virus (RSV) F, Ret, reticulon 4, Rheumatoidfactors, RLI P76, RPA2, RPK-1, RSK, RSV Fgp, S100, RON-8, SCF/KL, SCGF,Sclerostin, SDF-1, SDF1α, SDF1β, SERINE, Serum Amyloid P, Serum albumin,sFRP-3, Shh, Shiga like toxin II, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF,SMOH, SOD, SPARC, sphingosine 1-phosphate receptor 1, Staphylococcallipoteichoic acid, Stat, STEAP, STEAP-II, stem cell factor (SCF),streptokinase, superoxide dismutase, syndecan-1, TACE, TACI, TAG-72(tumor-associated glycoprotein-72), TARC, TB, TCA-3, T-cell receptoralpha/beta, TdT, TECK, TEM1, TEMS, TEM7, TEM8, Tenascin, TERT,testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha,TGF-beta, TGF-beta Pan Specific, TGF-beta RII, TGF-beta RIIb, TGF-betaRIII, TGF-beta R1 (ALK-5), TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4,TGF-beta5, TGF-I, Thrombin, thrombopoietin (TPO), Thymic stromallymphoprotein receptor, Thymus Ck-1, thyroid stimulating hormone (TSH),thyroxine, thyroxine-binding globulin, Tie, TIMP, TIQ, Tissue Factor,tissue factor protease inhibitor, tissue factor protein, TMEFF2, Tmpo,TMPRSS2, TNF receptor I, TNF receptor II, TNF-alpha, TNF-beta,TNF-beta2, TNFc, TNF-RT, TNF-RII, TNFRSF 10A (TRAIL R1 Apo-2/DR4),TNFRSF10B (TRAIL R2 DR5/KILLER/TRICK-2A/TRICK-B), TNFRSF10C (TRAIL R3DcRl/LIT/TRID), TNFRSF10D (TRAIL R4 DcR2/TRUNDD), TNFRSF11A (RANK ODFR/TRANCE R), TNFRSF11B (OPG OCIF/TR1), TNFRSF12 (TWEAK R FN14),TNFRSF12A, TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEMATAR/HveA/LIGHT R/TR2), TNFRSF 16 (NGFR p75NTR), TNFRSF 17 (BCMA),TNFRSF 18 (GITR ATTR), TNFRSF19 (TROY TAJ/TRADE), TNFRSF19L (RELT),TNFRSF1A (TNF R1 CD120a/p55-60), TNFRSF1B (TNF RII CD120b/p75-80),TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRSF25 (DR3Apo-3/LARD/TR-3/TRAMP/WSL-1), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNFRIII/TNFC R), TNFRSF4 (OX40 ACT35/TXGP1R), TNFRSF5 (CD40 p50), TNFRSF6(Fas Apo-1/APT1/CD95), TNFRSF6B (DcR3 M68/TR6), TNFRSF7 (CD27), TNFRSF8(CD30), TNFRSF9 (4-1 BB CD137/ILA), TNFRST23 (DcTRAIL R1 TNFRH1),TNFSF10 (TRAIL Apo-2 Ligand/TL2), TNFSF11 (TRANCE/RANK Ligand ODF/OPGLigand), TNFSF12 (TWEAK Apo-3 Ligand/DR3 Ligand), TNFSF13 (APRIL TALL2),TNFSF13B (BAFF BLYS/TALL1/THANK/TNFSF20), TNFSF14 (LIGHT HVEMLigand/LTg), TNFSF15 (TL1A/VEGI), TNFSF18 (GITR Ligand AITR Ligand/TL6),TNFSF1A (TNF-a Conectin/DIF/TNFSF2), TNFSF1B (TNF-b LTa/TNFSF1), TNFSF3(LTb TNFC/p33), TNFSF4 (OX40 Ligand gp34/TXGP1), TNFSF5 (CD40 LigandCD154/gp39/HIGM1/IMD3/TRAP), TNFSF6 (Fas Ligand Apo-1 Ligand/APT1Ligand), TNFSF7 (CD27 Ligand CD70), TNFSF8 (CD30 Ligand CD153), TNFSF9(4-1 BB Ligand CD137 Ligand), TNF-α, TNF-β, TNIL-1, toxic metabolite,TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrinreceptor, transforming growth factors (TGF) such as TGF-alpha andTGF-beta, Transmembrane glycoprotein NMB, Transthyretin, TRF, Trk,TROP-2, Trophoblast glycoprotein, TSG, TSLP, Tumor Necrosis Factor(TNF), tumor-associated antigen CA 125, tumor-associated antigenexpressing Lewis Y related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1,Urokinase, VAP-1, vascular endothelial growth factor (VEGF), vaspin,VCAM, VCAM-1, VECAD, VE-Cadherin, VE-Cadherin-2, VEFGR-1 (flt-1),VEFGR-2, VEGF receptor (VEGFR), VEGFR-3 (flt-4), VEGI, VIM, Viralantigens, VitB12 receptor, Vitronectin receptor, VLA, VLA-1, VLA-4, VNRintegrin, von Willebrand Factor (vWF), WIF-1, WNT1, WNT10A, WNT10B,WNT11, WNT16, WNT2, WNT2B/13, WNT3, WNT3A, WNT4, WNTSA, WNTSB, WNT6,WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, XCL1, XCL2/SCM-1-beta,XCL1/Lymphotactin, XCR1, XEDAR, XIAP, XPD.

One or more amino acid residue alterations are allowed in the amino acidsequences constituting the variable regions as long as theirantigen-binding activities are maintained. When altering a variableregion amino acid sequence, there is no particularly limitation on thesite of alteration and number of amino acids altered. For example, aminoacids present in CDR and/or FR can be altered appropriately. Whenaltering amino acids in a variable region, the binding activity ispreferably maintained without particular limitation; and for example, ascompared to before alteration, the binding activity is 50% or more,preferably 80% or more, and more preferably 100% or more. Furthermore,the binding activity may be increased by amino acid alterations. Forexample, the binding activity may be 2-, 5-, 10-times higher or suchthan that before alteration. In the antibodies of the present invention,alteration of amino acid sequence may be at least one of amino acidresidue substitution, addition, deletion, insertion, and modification.

For example, the modification of the N-terminal glutamine of a variableregion into pyroglutamic acid by pyroglutamylation is a modificationwell known to those skilled in the art. Thus, when the heavy-chain Nterminus is glutamine, the antibodies of the present invention comprisethe variable regions in which the glutamine is modified to pyroglutamicacid.

Antibody variable regions of the present invention may have anysequences, and they may be antibody variable regions of any origin, suchas mouse antibodies, rat antibodies, rabbit antibodies, goat antibodies,camel antibodies, humanized antibodies produced by humanizing thesenon-human antibodies, and human antibodies. “Humanized antibodies”, alsoreferred to as “reshaped human antibodies”, are antibodies in which thecomplementarity determining regions (CDRs) of an antibody derived from anon-human mammal, for example, a mouse antibody, are transplanted intothe CDRs of a human antibody. Methods for identifying CDRs are known(Kabat et al., Sequence of Proteins of Immunological Interest (1987),National Institute of Health, Bethesda, Md.; Chothia et al., Nature(1989) 342: 877). Their common genetic recombination technologies arealso known (see, European Patent Application Publication No. EP 125023and WO 96/02576). Furthermore, these antibodies may have various aminoacid substitutions introduced into their variable regions to improvetheir antigen binding, pharmacokinetics, stability, and antigenicity.Variable regions of the antibodies of the present invention may be ableto bind antigens repeatedly due to their pH dependability in antigenbinding (WO/2009/125825).

κ chain and λ chain-type constant regions are present in antibodylight-chain constant regions, but either one of the light chain constantregions is acceptable. Furthermore, light-chain constant regions of thepresent invention may be light-chain constant regions with amino acidalterations such as substitutions, additions, deletions, insertions,and/or modifications.

For example, for the heavy chain constant regions of an antibody of thepresent invention, heavy chain constant regions of human IgG antibodiesmay be used and heavy chain constant regions of human IgG1 antibodiesare preferred.

The variable regions constituting an antibody of the present inventioncan be variable regions that recognize any antigen. One or several aminoacid residues in the amino acid sequence constituting a heavy chainvariable region can be altered as long as the antigen-binding activityis maintained.

Furthermore, alteration of variable regions is carried out with theobjective of increasing binding activity, improving specificity,lowering pI, conferring a pH-dependent property to antigen binding,improving thermal stability of binding, improving solubility, providingstability to chemical modification, improving sugar-chain-derivedheterogeneity, avoiding T cell epitope that reduces immunogenicityidentified by in silico prediction, or by an in vitro assay using Tcells, introducing T cell epitope that activates regulatory T cells, orsuch (mAbs 3: 243-247, 2011).

Furthermore, a polypeptide of the present invention may be an Fc fusionprotein molecule produced by linking an Fc region with another protein,a biologically active peptide, or such (peptide Fc fusion protein), oran Fc fusion protein molecule produced by linking an Fc region with anextracellular matrix composed of polymers such as collagen or polylacticacid (scaffold Fc fusion protein).

Examples of another protein or biologically active peptide includereceptors, adhesion molecules, ligands, and enzymes, but are not limitedthereto.

Preferred examples of Fc fusion protein molecules of the presentinvention include proteins with Fc domain fused to a receptor proteinthat binds to a target, and such examples include TNFR-Fc fusionprotein, IL1R-Fc fusion protein, VEGFR-Fc fusion protein, and CTLA4-Fcfusion protein (Nat. Med. 2003 January; 9(1): 47-52; BioDrugs. 2006;20(3): 151-60). Furthermore, a protein to be fused to a polypeptide ofthe present invention may be any molecule as long as it binds to atarget molecule, and examples include scFv molecules (WO 2005/037989),single-domain antibody molecules (WO 2004/058821; WO 2003/002609),antibody-like molecules (Current Opinion in Biotechnology 2006, 17:653-658; Current Opinion in Biotechnology 2007, 18: 1-10; CurrentOpinion in Structural Biology 1997, 7: 463-469; and Protein Science2006, 15: 14-27) such as DARPins (WO 2002/020565), Affibody (WO1995/001937), Avimer (WO 2004/044011; WO 2005/040229), and Adnectin (WO2002/032925). Furthermore, antibodies and Fc fusion protein moleculesmay be multispecific antibodies that bind to multiple types of targetmolecules or epitopes such as bispecific antibodies.

Furthermore, the antibodies of the present invention include antibodymodification products. Such antibody modification products include, forexample, antibodies linked with various molecules such as polyethyleneglycol (PEG) and cytotoxic substances. Such antibody modificationproducts can be obtained by chemically modifying antibodies of thepresent invention. Methods for modifying antibodies are alreadyestablished in this field.

The antibodies of the present invention may also be bispecificantibodies. “Bispecific antibody” refers to an antibody that has in asingle antibody molecule variable regions that recognize differentepitopes. The epitopes may be present in a single molecule or indifferent molecules.

The polypeptides of the present invention can be prepared by the methodsknown to those skilled in the art. For example, the antibodies can beprepared by the methods described below, but the methods are not limitedthereto.

There are various known host cell/expression vector combinations forantibody preparation by introducing isolated genes encoding thepolypeptide into appropriate hosts. All of these expression systems areapplicable to isolation of the antigen-binding molecules of the presentinvention. Appropriate eukaryotic cells used as host cells includeanimal cells, plant cells, and fungal cells. Specifically, the animalcells include, for example, the following cells.

(1) mammalian cells: CHO (Chinese hamster ovary cell line), COS (Monkeykidney cell line), myeloma (Sp2/O, NS0 and such), BHK (baby hamsterkidney cell line), HEK293 (human embryonic kidney cell line with shearedadenovirus (Ad)5 DNA), PER.C6 cell (human embryonic retinal cell linetransformed with the Adenovirus Type 5 (Ad5) E1A and E1B genes), Hela,Vero, or such (Current Protocols in Protein Science (May, 2001, Unit5.9, Table 5.9.1));(2) amphibian cells: Xenopus oocytes, or such; and(3) insect cells: sf9, sf21, Tn5, or such.

A DNA encoding an antibody heavy chain in which one or more amino acidresidues in the Fc region have been substituted with other amino acidsof interest and DNA encoding an antibody light chain, are expressed. ADNA encoding a heavy chain in which one or more amino acid residues inthe Fc region are substituted with other amino acids of interest can beprepared, for example, by obtaining a DNA encoding the Fc region of anatural heavy chain, and introducing an appropriate substitution so thata codon encoding a particular amino acid in the Fc region encodesanother amino acid of interest.

Alternatively, a DNA encoding a heavy chain in which one or more aminoacid residues in the Fc region are substituted with other amino acids ofinterest can also be prepared by designing and then chemicallysynthesizing a DNA encoding a protein in which one or more amino acidresidues in the Fc region of the natural heavy chain are substitutedwith other amino acids of interest. The position and type of amino acidsubstitution are not particularly limited. Furthermore, alteration isnot limited to substitution, and alteration may be any of deletion,addition, or insertion, or combination thereof.

Alternatively, a DNA encoding a heavy chain in which one or more aminoacid residues in the Fc region are substituted with other amino acids ofinterest can be prepared as a combination of partial DNAs. Suchcombinations of partial DNAs include, for example, the combination of aDNA encoding a variable region and a DNA encoding a constant region, andthe combination of a DNA encoding an Fab region and a DNA encoding an Fcregion, but are not limited thereto. Furthermore, a DNA encoding a lightchain can similarly be prepared as a combination of partial DNAs.

Methods for expressing the above-described DNAs include the methodsdescribed below. For example, a heavy chain expression vector isconstructed by inserting a DNA encoding a heavy chain variable regioninto an expression vector along with a DNA encoding a heavy chainconstant region. Likewise, a light chain expression vector isconstructed by inserting a DNA encoding a light chain variable regioninto an expression vector along with a DNA encoding a light chainconstant region. Alternatively, these heavy and light chain genes may beinserted into a single vector.

When inserting a DNA encoding the antibody of interest into anexpression vector, the DNA is inserted so that the antibody is expressedunder the control of an expression-regulating region such as an enhanceror promoter. Next, host cells are transformed with this expressionvector to express the antibody. In such cases, an appropriatecombination of host and expression vector may be used.

Examples of the vectors include M13 vectors, pUC vectors, pBR322,pBluescript, and pCR-Script. Alternatively, when aiming to subclone andexcise cDNA, in addition to the vectors described above, pGEM-T,pDIRECT, pT7, and such can be used.

Expression vectors are particularly useful when using vectors forproducing the antibodies of the present invention. For example, when ahost cell is E. coli such as JM109, DH5α, HB101, and XL1-Blue, theexpression vectors must carry a promoter that allows efficientexpression in E. coli, for example, lacZ promoter (Ward et al., Nature(1989) 341: 544-546; FASEB J. (1992) δ: 2422-2427; its entirety areincorporated herein by reference), araB promoter (Better et al., Science(1988) 240: 1041-1043; its entirety are incorporated herein byreference), T7 promoter, or such. Such vectors include pGEX-5X-1(Pharmacia), “QIAexpress system” (QIAGEN), pEGFP, or pET (in this case,the host is preferably BL21 that expresses T7 RNA polymerase) inaddition to the vectors described above.

The vectors may contain signal sequences for polypeptide secretion. As asignal sequence for polypeptide secretion, a pelB signal sequence (Lei,S. P. et al J. Bacteriol. (1987) 169: 4379; its entirety areincorporated herein by reference) may be used when a polypeptide issecreted into the E. coli periplasm. The vector can be introduced intohost cells by lipofectin method, calcium phosphate method, andDEAE-Dextran method, for example.

In addition to E. coli expression vectors, the vectors for producing thepolypeptides of the present invention include mammalian expressionvectors (for example, pcDNA3 (Invitrogen), pEGF-BOS (Nucleic Acids. Res.1990, 18(17): p5322; its entirety are incorporated herein by reference),pEF, and pCDM8), insect cell-derived expression vectors (for example,the “Bac-to-BAC baculovirus expression system” (GIBCO BRL) andpBacPAK8), plant-derived expression vectors (for example, pMH1 andpMH2), animal virus-derived expression vectors (for example, pHSV, pMV,and pAdexLcw), retroviral expression vectors (for example, pZIPneo),yeast expression vectors (for example, “Pichia Expression Kit”(Invitrogen), pNV11, and SP-Q01), and Bacillus subtilis expressionvectors (for example, pPL608 and pKTH50), for example.

When aiming for expression in animal cells such as CHO, COS, NIH3T3, andHEK293 cells, the vectors must have a promoter essential for expressionin cells, for example, SV40 promoter (Mulligan et al., Nature (1979)277: 108; its entirety are incorporated herein by reference), MMTV-LTRpromoter, EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18:5322; its entirety are incorporated herein by reference), CAG promoter(Gene. (1990) 18: 5322; its entirety are incorporated herein byreference), and CMV promoter, and more preferably they have a gene forselecting transformed cells (for example, a drug resistance gene thatallows evaluation using an agent (neomycin, G418, or such)). Vectorswith such characteristics include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV,and pOP13, for example. Also, in some cases, the EBNA1 protein may befurther co-expressed for the purpose of increasing the gene copy number,and in this case, a vector having a replication initiation point OriP isused. (Biotechnol. Bioeng. 2001 Oct. 20; 75(2): 197-203; and Biotechnol.Bioeng. 2005 Sep. 20; 91(6): 670-7.)

In addition, the following method can be used for stable gene expressionand gene copy number amplification in cells: CHO cells deficient in anucleic acid synthesis pathway are introduced with a vector that carriesa DHFR gene which compensates for the deficiency (for example, pCHOI),and the vector is amplified using methotrexate (MTX). Alternatively, thefollowing method can be used for transient gene expression: COS cellswith a gene expressing SV40 T antigen on their chromosome aretransformed with a vector with an SV40 replication origin (pcD andsuch). Replication origins derived from polyoma virus, adenovirus,bovine papilloma virus (BPV), and such can also be used. To amplify genecopy number in host cells, the expression vectors may further carryselection markers such as aminoglycoside transferase (APH) gene,thymidine kinase (TK) gene, E. coli xanthine-guaninephosphoribosyltransferase (Ecogpt) gene, and dihydrofolate reductase(dhfr) gene.

Antibodies can be collected, for example, by culturing transformedcells, and then separating the antibodies from the inside of thetransformed cells or from the culture media. Antibodies can be separatedand purified using an appropriate combination of methods such ascentrifugation, ammonium sulfate fractionation, salting out,ultrafiltration, 1q, FcRn, protein A, protein G column, affinitychromatography, ion exchange chromatography, and gel filtrationchromatography.

As an efficient method for producing bispecific antibodies, theKnobs-into-holes technology may be used. Specifically, to produce aheterodimerized polypeptide of the present invention, it is necessary tohave association between polypeptides having amino acids that differfrom each other, or to separate the heterodimerized polypeptide ofinterest from the other homodimerized polypeptides.

For association of polypeptides having different amino acids from eachanother and comprising an Fc region, a technology of suppressingunintended association between H chains by introducing electrostaticrepulsion into the interface of the second constant region of theantibody H chain (CH2) or the third constant region of the H chain (CH3)(WO 2006/106905) can be applied.

In the technology of suppressing unintended association between H chainsby introducing electrostatic repulsion into the interface of CH2 or CH3,examples of amino acid residues in contact at the interface of otherconstant regions of the H chain include the residue at position 356 (EUnumbering), the residue at position 439 (EU numbering), the regionfacing the residue at position 357 (EU numbering), the residue atposition 370 (EU numbering), the residue at position 399 (EU numbering),and the residue at position 409 (EU numbering) in the CH3 domain.

More specifically, for example, in an antibody containing two types of Hchain CH3 domains, the antibody in which one to three pairs of aminoacid residues selected from the amino acid residues shown below in (1)to (3) in the first H chain CH3 domain have the same type of charge canbe produced:

-   (1) amino acid residues at positions 356 and 439 (EU numbering)    which are amino acid residues contained in the H chain CH3 domain;-   (2) amino acid residues at positions 357 and 370 (EU numbering)    which are amino acid residues contained in the H chain CH3 domain;    and-   (3) amino acid residues at positions 399 and 409 (EU numbering)    which are amino acid residues contained in the H chain CH3 domain.

Furthermore, an antibody can be produced in which one to three pairs ofamino acid residues corresponding to the amino acid residue pairsindicated above in (1) to (3) having the same type of charge in thefirst H chain CH3 domain have charges opposite to the correspondingamino acid residues in the aforementioned first H chain CH3 domain,wherein the amino acid residue pairs are selected from the amino acidresidue pairs indicated above in (1) to (3) in the second H chain CH3domain which differs from the first H chain CH3 domain.

The respective amino acid residues of (1) to (3) mentioned above arepositioned close to each other when associated. Those skilled in the artcan find sites that correspond to the above-mentioned amino acidresidues of (1) to (3) by homology modeling and such using commerciallyavailable software for the desired H chain CH3 domain or H chainconstant region, and amino acid residues of these sites can be alteredwhen appropriate.

In the above-mentioned antibodies, for example, “charged amino acidresidues” are preferably selected from amino acid residues included ineither of groups (X) or (Y) below:

(X) glutamic acid (E) and aspartic acid (D); and

(Y) lysine (K), arginine (R), and histidine (H).

In the above-mentioned antibodies, the phrase “having the same type ofcharge” means that, for example, all of the two or more amino acidresidues are amino acid residues included in either of theabove-mentioned groups (X) and (Y). The phrase “having the oppositecharge” means that, for example, when at least one of the two or moreamino acid residues is an amino acid residue included in either one ofthe above-mentioned groups (X) and (Y), the remaining amino acidresidues are amino acid residues included in the other group.

In a preferred embodiment of the above-mentioned antibody, the first Hchain CH3 domain and the second H chain CH3 domain may be cross-linkedby disulfide bonds.

In the present invention, the amino acid residues to be altered are notlimited to amino acid residues of the antibody constant region orantibody variable region described above. Those skilled in the art canfind amino acid residues that form the interface in polypeptide mutantsor heteromultimers through homology modeling and such using commerciallyavailable software, and can alter the amino acid residues at those sitesto regulate association.

Other known technologies can also be used for the association ofpolypeptides of the present invention having different amino acids andcomprising an Fc region. Polypeptides having different amino acids andcomprising an Fc region can be efficiently associated with each other bysubstituting an amino acid side chain present in one of the H chainvariable regions of the antibody with a larger side chain (knob), andsubstituting an amino acid side chain present in the opposing variableregion of the other H chain with a smaller side chain (hole), to allowplacement of the knob within the hole (WO 1996/027011; and Ridgway J Bet al., Protein Engineering (1996) 9, 617-621; Merchant A M et al.Nature Biotechnology (1998) 16, 677-681).

In addition, other known technologies can also be used for theassociation of polypeptides having different amino acids and comprisingan Fc region. Association of polypeptides having different sequences canbe induced efficiently by complementary association of CH3, by using astrand-exchange engineered domain CH3 produced by changing part of theCH3 in one of the H chains of an antibody into an IgA-derived sequencecorresponding to that portion, and introducing a correspondingIgA-derived sequence into the complementary portion of the CH3 on theother H chain (Protein Engineering Design & Selection, 23; 195-202,2010). This known technology can also be used to efficiently induceassociation of polypeptides having different amino acids and comprisingan Fc region.

In addition, one can also use technologies for heterodimerized antibodyproduction using association of antibody CH1 and CL, and association ofVH and VL, which are described in WO 2011/028952.

Furthermore, even in cases where heterodimerized polypeptides cannot beformed efficiently, heterodimerized polypeptides can be obtained byseparating and purifying them from homodimerized polypeptides. Whenproducing a heterodimerized polypeptide consisting of a firstpolypeptide and a second polypeptide which have different sequences fromeach other, homodimerized polypeptides consisting of only two firstpolypeptides, and homodimerized polypeptide consisting of only twosecond polypeptide are mixed in as impurities. Known technologies can beused as a method for efficiently removing these two types ofhomodimerized polypeptides. A method has been reported to be able topurify two types of homodimers and the heterodimerized antibody ofinterest by ion exchange chromatography, by creating a difference inisoelectric points by introducing amino acid substitutions into thevariable regions of the two types of H chains (WO 2007114325).

Regarding amino acid alteration for conferring difference in isoelectricpoints, the amino acid alteration to be introduced is not particularlylimited as long as a difference is produced between the isoelectricpoints of the two associating polypeptides, and it may also includeamino acid alterations made for other purposes such as loweringimmunogenicity. The altered amino acids are preferably amino acids atpositions with little influence on the binding activity towards an Fcγreceptor. Furthermore, it may be an amino acid alteration that increasesbinding activity to a desired Fcγ receptor. For such alterations, it ispreferable to introduce at least one amino acid mutation into an aminoacid position selected from specifically the group consisting of Gly atposition 137, Gly at position 138, Thr at position 139, Lys at position147, Ser at position 192, Leu at position 193, Gln at position 196, Tyrat position 198, Ile at position 199, Asn at position 203, Lys atposition 214, Val at position 263, Glu at position 272, Lys at position274, Tyr at position 278, Lys at position 288, Lys at position 290, Glyat position 316, Lys at position 317, Lys at position 320, Lys atposition 324, Thr at position 335, Ser at position 337, Lys at position340, Leu at position 358, Lys at position 360, Gln at position 362, Serat position 364, Ser at position 383, Asn at position 384, Gly atposition 385, Gln at position 386, Pro at position 387, Asn at position390, Val at position 397, and Val at position 422 (EU numbering) in theamino acid sequence of the first polypeptide and/or the secondpolypeptide. Furthermore, it is preferable to introduce a mutation intoat least one amino acid selected from the group consisting of Gly atposition 137, Gly at position 138, Thr at position 139, Lys at position147, Ser at position 192, Leu at position 193, Gln at position 196, Ileat position 199, Asn at position 203, Lys at position 214, Glu atposition 272, Lys at position 274, Lys at position 288, Lys at position290, Leu at position 358, Lys at position 360, Gln at position 362, Serat position 383, Asn at position 384, Gly at position 385, Gln atposition 386, Asn at position 390, Val at position 397, and Val atposition 422 (EU numbering). Further, it is more preferable to introducea mutation into at least one amino acid selected from the groupconsisting of Gly at position 137, Gly at position 138, Lys at position147, Ser at position 192, Leu at position 193, Gln at position 196, Ileat position 199, Asn at position 203, Lys at position 214, Lys atposition 274, Lys at position 288, Leu at position 358, Asn at position384, and Val at position 397 (EU numbering).

More specifically, it is preferable that a mutation is introduced intoat least one amino acid selected from the group consisting of Gln atposition 196, Ile at position 199, Val at position 263, Glu at position272, Gly at position 316, Leu at position 358, Ser at position 364, Serat position 383, Pro at position 387, and Val at position 397 (EUnumbering) in the amino acid sequence of either polypeptide of the firstpolypeptide and the second polypeptide; and preferably a mutation isintroduced into at least one amino acid selected from the groupconsisting of Gly at position 137, Gly at position 138, Thr at position139, Lys at position 147, Ser at position 192, Leu at position 193, Tyrat position 198, Ile at position 199, Asn at position 203, Lys atposition 214, Lys at position 274, Tyr at position 278, Lys at position288, Lys at position 290, Gly at position 316, Lys at position 317, Lysat position 320, Lys at position 324, Thr at position 335, Ser atposition 337, Lys at position 340, Leu at position 358, Lys at position360, Gln at position 362, Ser at position 383, Asn at position 384, Glyat position 385, Gln at position 386, Asn at position 390, and Val atposition 422 (EU numbering) in the amino acid sequence of the otherpolypeptide. Furthermore, it is preferable that a mutation is introducedinto at least one amino acid selected from the group consisting of Glnat position 196, Ile at position 199, Glu at position 272, Leu atposition 358, Ser at position 383, and Val at position 397 (EUnumbering) in the amino acid sequence of one of the polypeptides; andpreferably a mutation is introduced into at least one amino acidselected from the group consisting of Gly at position 137, Gly atposition 138, Thr at position 139, Lys at position 147, Ser at position192, Leu at position 193, Ile at position 199, Asn at position 203, Lysat position 214, Lys at position 274, Lys at position 288, Lys atposition 290, Leu at position 358, Lys at position 360, Gln at position362, Ser at position 383, Asn at position 384, Gly at position 385, Glnat position 386, Asn at position 390, and Val at position 422 (EUnumbering) in the amino acid sequence of the other polypeptide.Furthermore, it is more preferable that a mutation is introduced into atleast one amino acid selected from the group consisting of Gln atposition 196, Ile at position 199, Leu at position 358, and Val atposition 397 (EU numbering) in the amino acid sequence of one of thepolypeptides; and that a mutation is introduced into at least one aminoacid selected from the group consisting of Gly at position 137, Gly atposition 138, Lys at position 147, Ser at position 192, Leu at position193, Ile at position 199, Asn at position 203, Lys at position 214, Lysat position 274, Lys at position 288, and Asn at position 384 (EUnumbering) in the amino acid sequence of the other polypeptide.

The amino acid alteration is not particularly limited as long as thealteration is made to produce a difference in isoelectric points betweenthe two associating polypeptides after the alteration.

Examples of a preferred alteration for increasing the isoelectric pointinclude substitution of the amino acid at position 196 with Lys,substitution of the amino acid at position 263 with Lys, substitution ofthe amino acid at position 272 with Lys, substitution of the amino acidat position 316 with Lys, substitution of the amino acid at position 364with Lys, substitution of the amino acid at position 358 with Lys,substitution of the amino acid at position 383 with Lys, substitution ofthe amino acid at position 387 with Lys, and substitution of the aminoacid at position 397 with Lys (EU numbering). Examples of a preferredalteration for decreasing the isoelectric point include substitution ofthe amino acid at position 137 with Glu, substitution of the amino acidat position 138 with Glu, substitution of the amino acid at position 139with Glu, substitution of the amino acid at position 147 with Glu,substitution of the amino acid at position 198 with Glu, substitution ofthe amino acid at position 203 with Asp, substitution of the amino acidat position 214 with Thr, substitution of the amino acid at position 274with Gln, substitution of the amino acid at position 278 with Glu,substitution of the amino acid at position 288 with Glu, substitution ofthe amino acid at position 290 with Glu, substitution of the amino acidat position 316 with Glu, substitution of the amino acid at position 317with Glu, substitution of the amino acid at position 320 with Glu,substitution of the amino acid at position 324 with Glu, substitution ofthe amino acid at position 335 with Glu, substitution of the amino acidat position 337 with Asp, substitution of the amino acid at position 340with Glu, substitution of the amino acid at position 358 with Glu,substitution of the amino acid at position 360 with Glu, substitution ofthe amino acid at position 362 with Glu, substitution of the amino acidat position 383 with Glu, substitution of the amino acid at position 384with Glu, substitution of the amino acid at position 385 with Glu,substitution of the amino acid at position 386 with Glu, substitution ofthe amino acid at position 390 with Glu, and substitution of the aminoacid at position 422 with Glu (EU numbering).

When combining amino acid alterations that are made for a purpose otherthan producing a difference in isoelectric points, for example, to lowerantigenicity, substitution of the amino acid at position 138 with Ser,substitution of the amino acid at position 192 with Asn, substitution ofthe amino acid at position 193 with Phe, and substitution of the aminoacid at position 199 with Thr (EU numbering) may be combined.

So far, as a method for purifying a heterodimerized antibody, a methodthat uses protein A for purifying a heterodimerized antibody comprisinga mouse IgG2a H chain that binds to protein A and a rat IgG2b H chainthat does not bind to protein A has been reported (WO 98050431 and WO95033844).

Furthermore, a heterodimerized antibody alone can be efficientlypurified by using H chains in which the amino acid residues at positions435 and 436 (EU numbering), which are the binding site between IgG andProtein A, are substituted with amino acids such as Tyr and His whichhave different affinity to Protein A to change the interaction betweeneach of the H chains and Protein A, and by using a Protein A column. Aplurality, for example, two or more, of these substitutions andtechnologies can be used in combination. Furthermore, when appropriate,these alterations can be applied separately to the first polypeptide andthe second polypeptide. Polypeptides of the present invention may bethose produced based on the polypeptides to which the above-mentionedalterations have been applied.

The present invention also provides a method for producing a polypeptidecomprising an Fc region, which comprises the steps of heterodimerizingthe Fc region by introducing an amino acid mutation into the firstpolypeptide and/or the second polypeptide constituting the Fc region,and introducing an amino acid mutation to alter the Fc region functioncompared to when the Fc region forms a homodimer.

Examples include a production method comprising the following steps of:

-   (a) in a polypeptide comprising an Fc region, introducing an amino    acid mutation into the first polypeptide and/or the second    polypeptide constituting the Fc region;-   (b) determining the Fc region function of the heterodimer consisting    of the first polypeptide and second polypeptide into which a    mutation is introduced in step (a); and-   (c) selecting a polypeptide with altered Fc region function compared    to the parent polypeptide or compared to when the Fc region is    homodimerized by introduction of the amino acid mutation.

In this production method, the following step may be performed afterstep (a):

-   (d) displaying the Fc region-containing heterodimerized polypeptide    consisting of the first polypeptide and the second polypeptide on    the presented ribosomes, phages, or yeast.

A preferred embodiment is a method for producing a polypeptidecomprising an Fc region which comprises the steps of:

-   (a) altering a nucleic acid encoding the polypeptide so that the Fc    region function is altered compared to the parent polypeptide or    compared to when the Fc region forms a homodimer by introduction of    amino acid mutation;-   (b) introducing the nucleic acid into a host cell and culturing the    cell to express the polypeptide; and-   (c) collecting the polypeptide from a host cell culture.

Antibodies and Fc fusion protein molecules produced by the productionmethod are also included in the present invention.

The type and range of amino acid mutations introduced by the presentmethod are not particularly limited, but examples include amino acidmutations involved in alteration of each Fc region function describedherein (more specifically, amino acid mutations disclosed specificallyin the Tables in the Examples).

The present invention also provides a method for altering the functionof a polypeptide comprising an Fc region, which comprises the steps ofheterodimerizing the Fc region by introducing an amino acid mutationinto the first polypeptide and/or the second polypeptide constitutingthe Fc region to alter the Fc region function compared to when the Fcregion forms a homodimer by introduction of the amino acid mutation.

Examples include alteration methods comprising the following steps of:

-   (a) in a polypeptide comprising an Fc region, introducing an amino    acid mutation into the first polypeptide and/or the second    polypeptide constituting the Fc region;-   (b) determining the Fc region function of the heterodimer consisting    of the first polypeptide and second polypeptide into which a    mutation is introduced in step (a); and-   (c) selecting a polypeptide with altered Fc region function compared    to the parent polypeptide or compared to when the Fc region is    homodimerized by introduction of the amino acid mutation.

In this alteration method, the following step may be performed afterstep (a):

-   (d) displaying the Fc region-containing heterodimerized polypeptide    consisting of the first polypeptide and the second polypeptide    presented on ribosomes, phages, or yeast.

A preferred embodiment is a method for altering a polypeptide comprisingan Fc region which comprises the steps of:

-   (a) altering a nucleic acid encoding the polypeptide so that the Fc    region function is altered compared to the parent polypeptide or    compared to when the Fc region forms a homodimer by introduction of    the amino acid mutation;-   (b) introducing the nucleic acid into a host cell and culturing the    cell to express the polypeptide; and-   (c) collecting the polypeptide from a host cell culture.

Antibodies and Fc fusion protein molecules altered by the alterationmethod are also included in the present invention.

The type and range of amino acid mutations introduced by the presentmethod are not particularly limited, but examples include amino acidmutations involved in alteration of each Fc region function describedherein (more specifically, amino acid mutations disclosed specificallyin the Tables in the Examples).

Furthermore, the present invention provides a nucleic acid encoding apolypeptide comprising an Fc region, wherein the polypeptide ischaracterized in that the Fc region is composed of a heterodimercomprising a first polypeptide and a second polypeptide, and wherein thepolypeptide is characterized in that an Fc region function is alteredcompared to that of a polypeptide characterized in that the Fc region iscomposed of a homodimer comprising only the first polypeptide and/orcompared to that of a polypeptide characterized in that the Fc region iscomposed of a homodimer comprising only the second polypeptide. Thenucleic acid of the present invention may be in any form such as DNA orRNA.

The present invention also provides vectors carrying the above-describednucleic acids of the present invention. The type of vector can beappropriately selected by those skilled in the art depending on the hostcells to be introduced with the vector. The vectors include, forexample, those described above.

Furthermore, the present invention relates to host cells transformedwith the above-described vectors of the present invention. Appropriatehost cells can be selected by those skilled in the art. The host cellsinclude, for example, those described above.

<Pharmaceutical Compositions>

The present invention provides pharmaceutical compositions comprisingthe polypeptide of the present invention.

The pharmaceutical compositions of the present invention can beformulated, in addition to the antibodies or Fc-fusion proteinmolecules, which are the polypeptides of the present invention, withpharmaceutically acceptable carriers by known methods. For example, thecompositions can be used parenterally, when the antibodies or Fc-fusionprotein molecules are formulated in a sterile solution or suspension forinjection using water or any other pharmaceutically acceptable liquid.For example, the compositions can be formulated by appropriatelycombining the antibodies or Fc-fusion protein molecules withpharmacologically acceptable carriers or media, specifically, sterilewater or physiological saline, vegetable oils, emulsifiers, suspendingagents, surfactants, stabilizers, flavoring agents, excipients,vehicles, preservatives, binding agents, and such, by mixing them at aunit dose and form required by generally accepted pharmaceuticalimplementations. Specific examples of the carriers include lightanhydrous silicic acid, lactose, crystalline cellulose, mannitol,starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose,hydroxypropyl methylcellulose, polyvinylacetal diethylaminoacetate,polyvinylpyrrolidone, gelatin, medium-chain triglyceride,polyoxyethylene hardened castor oil 60, saccharose, carboxymethylcellulose, corn starch, inorganic salt, and such. The content of theactive ingredient in such a formulation is adjusted so that anappropriate dose within the required range can be obtained.

Sterile compositions for injection can be formulated using vehicles suchas distilled water for injection, according to standard protocols.

Aqueous solutions used for injection include, for example, physiologicalsaline and isotonic solutions containing glucose or other adjuvants suchas D-sorbitol, D-mannose, D-mannitol, and sodium chloride. These can beused in conjunction with suitable solubilizers such as alcohol,specifically ethanol, polyalcohols such as propylene glycol andpolyethylene glycol, and non-ionic surfactants such as Polysorbate 80™and HCO-50.

Oils include sesame oils and soybean oils, and can be combined withsolubilizers such as benzyl benzoate or benzyl alcohol. These may alsobe formulated with buffers, for example, phosphate buffers or sodiumacetate buffers; analgesics, for example, procaine hydrochloride;stabilizers, for example, benzyl alcohol or phenol; or antioxidants. Theprepared injections are typically aliquoted into appropriate ampules.

The administration is preferably carried out parenterally, andspecifically includes injection, intranasal administration,intrapulmonary administration, and percutaneous administration. Forexample, injections can be administered systemically or locally byintravenous injection, intramuscular injection, intraperitonealinjection, or subcutaneous injection.

Furthermore, the method of administration can be appropriately selectedaccording to the age and symptoms of the patient. A single dosage of thepharmaceutical composition containing a polypeptide or a polynucleotideencoding a polypeptide can be selected, for example, from the range of0.0001 to 1,000 mg per kg of body weight. Alternatively, the dosage maybe, for example, in the range of 0.001 to 100,000 mg/patient. However,the dosage is not limited to these values. The dosage and method ofadministration vary depending on the patient's body weight, age, andsymptoms, and can be appropriately selected by those skilled in the art.

In the present invention, the pharmaceutical compositions comprising thepolypeptides of the present invention described above are useful asactive ingredients for therapeutic agents or preventive agents forcancer, immune inflammatory diseases, and such.

As used herein, the three-letter and single-letter codes for respectiveamino acids are as follows:

Alanine: Ala (A)

Arginine: Arg (R)

Asparagine: Asn (N)

Aspartic acid: Asp (D)

Cysteine: Cys (C)

Glutamine: Gln (O)

Glutamic acid: Glu (E)

Glycine: Gly (G)

Histidine: His (H)

Isoleucine: Ile (I)

Leucine: Leu (L)

Lysine: Lys (K)

Methionine: Met (M)

Phenylalanine: Phe (F)

Proline: Pro (P)

Serine: Ser (S)

Threonine: Thr (T)

Tryptophan: Trp (W)

Tyrosine: Tyr (Y)

Valine: Val (V)

All prior art documents cited herein are incorporated by reference intheir entirety.

EXAMPLES

Herein below, the present invention will be specifically describedfurther with reference to the Examples, but it is not to be construed asbeing limited thereto.

[Example 1] Explanation of the Concept of the Improvement of FcγRRecognition by Heterodimerized Antibodies

An antibody interacts via its Fc region with various molecules such asFcRn, FcγR, and complements. A single molecule of FcRn, which is aligand of Fc, binds to each one of the heavy chains (H chains) of anantibody. Thus, two molecules of FcRn bind to a single antibody molecule(FIG. 1). In vivo, FcRn is expressed on the cell membrane. Thus, anantibody recognizes two molecules of FcRn in a symmetrical manner viathe identical sites in its respective H chains in vivo (Nature, 372:379-383, 1994). Furthermore, in a manner similar to the relationshipbetween IgG and FcRn, a single molecule of IgA, which belongs to thesame immunoglobulin family as IgG, recognizes in a symmetrical mannertwo molecules of FcαR, which is an IgA receptor (FIG. 2) (Nature, 423:614-620, 2003).

However, unlike FcRn and others, only one molecule of FcγR binds to onemolecule of an antibody (FIG. 3) (JBC, 276: 16469-16477, 2001). IgGrecognizes FcγR via the CH2 domains of the two H chains; however, theFcγR-interaction sites are different between the two H chains. Forexample, when the H chain shown at the left side of FIG. 3 is defined asH_(A) chain, and the one at the right side as H_(B) chain, Ala atposition 327 (EU numbering) in each of the H_(A) and H_(B) chainsinteracts with FcγR. However, there is a difference between theproperties of the partner residues with which the respective H chainsinteract (FIG. 4). The H_(A) chain interacts with FcγRIII in ahydrophobic manner at Trp of positions 87 and 110 (EU numbering), whilethe H_(B) chain interacts with FcγRIII at His of position 131 (EUnumbering). Hence, when Ala at position 327 (EU numbering) issubstituted with a highly-hydrophobic amino acid such as Trp, it canreduce the FcγR-binding activity of H_(B) chain even if it has theeffect of improving the FcγR-binding activity of H_(A) chain. Thus, theasymmetric effect of the two H chains on FcγR needs to be considered tooptimize the interaction of the Fc region of IgG with FcγR by amino acidalteration. Nevertheless, in the prior art, the same alteration has beenintroduced into the two H chains to optimize the interaction of the Fcregion of IgG with FcγR (WO 2006/019447 and WO 2000/042072). However,when considering the asymmetric interaction the Fc region of IgG withFcγR, the interaction between IgG and FcγR can be optimized more finelyby introducing different alterations into the H chains. That is, theinteraction with FcγR can be more finely optimized by using aheterodimerized antibody resulting from the introduction of differentalterations into the two H chains to optimize the interaction of the Fcregion with FcγR as compared to a homodimerized antibody resulting fromthe introduction of the same alteration into the H chains, which hasbeen performed in the prior art.

[Example 2] Proof of the Concept of the Improvement of FcγR Recognitionby Heterodimerized Antibodies

It was assessed whether the FcγR-binding activity of an antibody can beoptimized more finely by using a heterodimerized antibody introducedwith different alterations into the two H chains as compared to ahomodimerized antibody of the prior art.

Conventionally, alterations that enhance the FcγR binding have beensought by using a homodimerized antibody resulting from the introductionof the same alteration into both H chains of an antibody. However, asdescribed in Example 1, an antibody interacts with FcγR in an asymmetricmanner, and when the same alteration is introduced into the two Hchains, the alteration in one H chain could enhance the FcγR-bindingactivity while the alteration in the other H chain could rather inhibitthe binding. The FcγR-binding activity is not necessarily increased in ahomodimerized antibody resulting from the introduction of such analteration into both H chains. However, a heterodimerized antibodyresulting from the introduction of the alteration into only one of thetwo H chains can have increased FcγR-binding activity.

In order to test this hypothesis, with respect to FcγR binding, aheterodimerized antibody comprising a first polypeptide in which onlyone H chain has been introduced with an alteration that is thought toalter the FcγR-binding activity and a second polypeptide without theabove alteration was compared with a homodimerized antibody comprisingthe first polypeptide in which only one H chain has been introduced withthe alteration that is thought to alter the FcγR-binding activity. Basedon the previous concept, when the FcγR-binding activity is increased bythe alteration, the homodimerized antibody is always superior to theheterodimerized antibody. However, if an antibody Fc recognizes FcγR inan asymmetric manner, the heterodimerized antibody is expected to showgreater FcγR-binding activity than the homodimerized antibody dependingon the type of alteration.

The H chain variable region of an antibody used was the variable regionof an anti-glypican-3 antibody which contains CDR of pH7 of theanti-glypican-3 antibody with improved kinetics in plasma disclosed inWO 2009/041062. The variable region is named GpH7 (SEQ ID NO: 1). Theconstant regions of antibody H chains described below were used incombination with GpH7. When the H chain constant region of an antibodyis named H1, the sequence of the antibody H chain having the variableregion GpH7 is referred to as GpH7-H1 Amino acid alterations areindicated in a manner such as D356K. The first alphabetical letter (forexample, “D” of D356K) is a one-letter code representing the amino acidresidue before alteration, and the following numeral (for example, “356”of D356K) indicates the position of alteration (EU numbering). The lastalphabetical letter (for example, “K” of D356K) is a one-letter coderepresenting the amino acid residue after alteration. GpH7-G1d (SEQ IDNO: 2) resulting from the removal of the C-terminal Gly and Lys from anIgG1 having the variable region GpH7; GpH7-A5 (SEQ ID NO: 3) resultingfrom the introduction of mutations D356K and H435R into GpH7-G1d; andGpH7-B3 (SEQ ID NO: 4) resulting from the introduction of K439E intoGpH7-G1d were prepared according to the method described in ReferenceExample 1. The mutations D356K and K439E were introduced into each Hchain to allow effective formation of heterodimer of the respective Hchains when producing a heterodimerized antibody comprising two types ofH chains (WO 2006/106905). H435R, which is an alteration that inhibitsProtein A binding, was introduced to allow efficient separation of theheterodimerized and homodimerized forms (see Reference Examples 3, 4,and 5). Meanwhile, the antibody L chain used was GpL16-k0 (SEQ ID NO:5), which is the L chain of the glypican-3 antibody with improvedkinetics in plasma disclosed in WO 2009/041062.

Mutations for proving the concept of the heterodimerized antibody wereintroduced into parental polypeptides GpH7-A5 and GpH7-B3 to constructmodified variants, and the variants were assessed. The constructedexpression vectors were used to transfect FreeStyle293 cells(Invitrogen) according to the method described in Reference Example 1.The expressed antibodies were purified according to the method describedin Reference Example 1. When expressing a homodimerized antibody, anexpression vector inserted with the antibody L chain GpL16-k0 was usedtogether with an expression vector inserted with one type of antibody Hchain sequence. When a heterodimerized antibody was expressed, anexpression vector inserted with the antibody L chain GpL16-k0, as usedfor the above homodimerized antibody, and an expression vector insertedwith a sequence resulting from the introduction of an additionalalteration into GpH7-A5 having the alteration D356K as one antibody Hchain, and an expression vector inserted with a sequence resulting fromthe introduction of an additional alteration into GpH7-B3 having thealteration K439E as the other antibody H chain, were used to achieveefficient expression of the heterodimerized antibody. The antibodyexpressed and purified is referred to, for example, asGpH7-H1/GpH7-H2/GpL16-k0, when the expression vector used to express oneantibody H chain of the heterodimerized antibody is GpH7-H1, theexpression vector for the other antibody H chain is GpH7-H2, and theexpression vector for the antibody L chain is GpL16-k0. In this system,a sequence introduced with the alterations D356K and H435R correspondsto H1, and a sequence introduced with the alteration K439E correspondsto H2. For example, when the expression vectors used to express theantibody H chain and L chain of a homodimerized antibody arerespectively GpH7-H1 and GpL16-k0, the homodimerized antibody isreferred to as GpH7-H1/GpL16-k0. Prepared antibodies were used tomeasure the FcγR-binding activity by the method described in ReferenceExample 2.

First, it was assessed whether the alterations D356K and H435Rintroduced into GpH7-A5, and the alteration K439E introduced intoGpH7-B3 to form and purify heterodimers had an effect on theFcγR-binding activity as compared to native IgG. As a control,GpH7-G1d/GpL16-k0 (SEQ ID NOs: 2 and 5, respectively) was expressedusing plasmids inserted with GpH7-G1d and GpL16-k0 as the antibody Hchain and L chain, respectively, and purified according to the method ofReference Example 1. Likewise, the homodimerized antibodyGpH7-A5/GpL16-k0 (SEQ ID NOs: 3 and 5, respectively) whose two H chainswere introduced with D356K and H435R, the homodimerized antibodyGpH7-B3/GpL16-k0 (SEQ ID NOs: 4 and 5, respectively) whose two H chainswere introduced with K439E, and the heterodimerized antibodyGpH7-A5/GpH7-B3/GpL16-k0 (SEQ ID NOs: 3, 4, and 5, respectively) inwhich one of the H chains was introduced with D356K and H435R and theother was introduced with K439E, were prepared. These antibodies andtheir binding activity to each FcγR were compared according to themethod described in Reference Example 2, and the result is summarized inFIG. 5.

The measurement result showed that there was no significant differencein the binding activity to each FcγR between GpH7-G1d/GpL16-k0 andGpH7-A5/GpH7-B3/GpL16-k0. Furthermore, with respect to each FcγR,GpH7-A5/GpL16-k0 and GpH7-B3/GpL16-k0 retained at least about 80% of thebinding activity of GpH7-G1d/GpL16-k0. Based on the above result, it wasdetermined that the FcγR binding of GpH7-A5/GpH7-B3/GpL16-k0,GpH7-A5/GpL16-k0, and GpH7-B3/GpL16-k0 was not significantly reduced ascompared to GpH7-G1d/GpL16-k0, and thus, variants resulting from theintroduction of mutations into each H chain of these antibodies can becompared for the binding activity to each FcγR.

Then, GpH7-A26 (SEQ ID NO: 6) resulting from the introduction ofmutation G237A into GpH7-A5 was constructed according to the methoddescribed in Reference Example 1. Using GpL16-k0 as the L chain, andGpH7-A26 and GpH7-B3 as the H chain, the heterodimerized antibodyGpH7-A26/GpH7-B3/GpL16-k0 (SEQ ID NOs: 6, 4, and 5, respectively) inwhich only one of the H chains has been introduced with G237A wasexpressed according to the method described in Reference Example 1.Likewise, using GpH7-A26 as the H chain and GpL16-k0 as the L chain, thehomodimerized antibody GpH7-A26/GpL16-k0 (SEQ ID NOs: 6 and 5,respectively) whose two H chains were introduced with G237A wasexpressed according to the method described in Reference Example 1.These antibodies were assessed for the binding activity to each FcγRaccording to the method described in Reference Example 2 (FIG. 6). Theresult showed that the heterodimerized antibodyGpH7-A26/GpH7-B3/GpL16-k0 had increased binding activities to FcγRIIa Rand FcγRIIb as compared to GpH7-A5/GpH7-B3/GpL16-k0. Meanwhile, thehomodimerized antibody GpH7-A26/GpL16-k0 in which the same alterationwas introduced into both H chains had reduced binding activities toFcγRIIa R and FcγRIIb as compared to GpH7-A5/GpH7-B3/GpL16-k0. Theseresults demonstrate that, G237A is an alteration that increases thebinding activities to FcγRIIa R and FcγRIIb when introduced into onlyone H chain, while reduces the binding activities to FcγRIIa R andFcγRIIb when introduced into both H chains.

Then, GpH7-A29 (SEQ ID NO: 7) resulting from the introduction of themutation G237L into GpH7-A5 was constructed according to the methoddescribed in Reference Example 1. Using GpL16-k0 as the L chain, andGpH7-A29 and GpH7-B3 as the H chain, the heterodimerized antibodyGpH7-A29/GpH7-B3/GpL16-k0 (SEQ ID NOs: 7, 4, and 5, respectively) inwhich only one of the H chains has been introduced with G237L wasexpressed according to the method described in Reference Example 1.Likewise, using GpH7-A29 as the H chain and GpL16-k0 as the L chain, thehomodimerized antibody GpH7-A29/GpL16-k0 (SEQ ID NOs: 7 and 5,respectively) whose two H chains were introduced with G237L wasexpressed according to the method described in Reference Example 1.These antibodies and their binding activities to each FcγR were assessedaccording to the method described in Reference Example 2 (FIG. 7). Theheterodimerized antibody GpH7-A29/GpH7-B3/GpL16-k0 had increased FcγRIIaR-binding and FcγRIIb-binding activities as compared toGpH7-A5/GpH7-B3/GpL16-k0. Meanwhile, the homodimerized antibodyGpH7-A29/GpL16-k0 whose two H chains had the same alteration had reducedbinding activity to FcγRIIa R and FcγRIIb as compared toGpH7-A5/GpH7-B3/GpL16-k0. These results demonstrate that, G237L is analteration that has an effect of increasing the binding activities toFcγRIIa R and FcγRIIb when introduced into only one H chain, whilereducing the binding activities to FcγRIIa R and FcγRIIb when introducedinto both H chains.

Then, GpH7-A42 (SEQ ID NO: 8) resulting from the introduction of themutation L328E into GpH7-A5 was constructed according to the methoddescribed in Reference Example 1. Using GpL16-k0 as the L chain, andGpH7-A42 and GpH7-B3 as the H chain, the heterodimerized antibodyGpH7-A42/GpH7-B3/GpL16-k0 (SEQ ID NOs: 8, 4, and 5, respectively) inwhich only one of the H chains has been introduced with L328E wasexpressed according to the method described in Reference Example 1.Likewise, using GpH7-A42 as the H chain and GpL16-k0 as the L chain, thehomodimerized antibody GpH7-A42/GpL16-k0 (SEQ ID NO: 8 and 5,respectively) whose two H chains were introduced with L328E wasexpressed according to the method described in Reference Example 1.These antibodies were assessed for the binding activity to each FcγRaccording to the method described in Reference Example 2 (FIG. 8). Theheterodimerized antibody GpH7-A42/GpH7-B3/GpL16-k0 had increased bindingactivity to FcγRIIa R and FcγRIIb as compared toGpH7-A5/GpH7-B3/GpL16-k0. Meanwhile, the homodimerized antibodyGpH7-A42/GpL16-k0 whose two H chains were introduced with the samealteration had reduced FcγRIIa R-binding activity and increasedFcγRIIb-binding activity as compared to GpH7-A5/GpH7-B3/GpL16-k0;however, the degree of increase was greater in GpH7-A42/GpH7-B3/GpL16-k0in which only one of the H chains was introduced with L328E. Theseresults demonstrate that L328E is an alteration that is more effectiveto increase the binding activities to FcγRIIa R and FcγRIIb whenintroduced into only one H chain than when introduced into both Hchains.

Then, GpH7-A43 (SEQ ID NO: 9) resulting from the introduction of themutation L328D into GpH7-A5 was constructed according to the methoddescribed in Reference Example 1. Using GpL16-k0 as the L chain, andGpH7-A43 and GpH7-B3 as the H chain, the heterodimerized antibodyGpH7-A43/GpH7-B3/GpL16-k0 (SEQ ID NOs: 9, 4, and 5, respectively) inwhich only one of the H chains has been introduced with L328D wasexpressed according to the method described in Reference Example 1.Likewise, using GpH7-A43 as the H chain and GpL16-k0 as the L chain, thehomodimerized antibody GpH7-A43/GpL16-k0 (SEQ ID NOs: 9 and 5,respectively) whose two H chains were introduced with L328D wasexpressed according to the method described in Reference Example 1.These antibodies were assessed for the binding activity to each FcγRaccording to the method described in Reference Example 2 (FIG. 9). Theheterodimerized antibody GpH7-A43/GpH7-B3/GpL16-k0 had increased bindingactivity to FcγRIIa R and FcγRIIb as compared toGpH7-A5/GpH7-B3/GpL16-k0. Meanwhile, the homodimerized antibodyGpH7-A43/GpL16-k0 whose two H chains were introduced with the samealteration had reduced FcγRIIa R-binding activity and increasedFcγRIIb-binding activity as compared to GpH7-A5/GpH7-B3/GpL16-k0;however, the degree of increase was greater in GpH7-A43/GpH7-B3/GpL16-k0in which only one of the H chains was introduced with L328D. Theseresults demonstrate that L328D is an alteration that is more effectiveto increase the binding activities to FcγRIIa R and FcγRIIb whenintroduced into only one H chain than when introduced into both Hchains.

Then, GpH7-B16 (SEQ ID NO: 10) resulting from the introduction of themutation L234E into GpH7-B3 was constructed according to the methoddescribed in Reference Example 1. Using GpL16-k0 as the L chain, andGpH7-A5 and GpH7-B16 as the H chain, the heterodimerized antibodyGpH7-A5/GpH7-B16/GpL16-k0 (SEQ ID NOs: 3, 10, and 5, respectively) inwhich only one of the H chains has been introduced with L234E wasexpressed according to the method described in Reference Example 1.Likewise, using GpH7-B16 as the H chain and GpL16-k0 as the L chain, thehomodimerized antibody GpH7-B16/GpL16-k0 (SEQ ID NOs: 10 and 5,respectively) whose two H chains were introduced with L234E wasexpressed according to the method described in Reference Example 1.These antibodies were assessed for the binding activity to each FcγRaccording to the method described in Reference Example 2 (FIG. 10). Theheterodimerized antibody GpH7-A5/GpH7-B16/GpL16-k0 had increased bindingactivities to FcγRIIIa F and FcγRIIb as compared toGpH7-A5/GpH7-B3/GpL16-k0. Meanwhile, the homodimerized antibodyGpH7-B16/GpL16-k0 whose two H chains were introduced with the samealteration had reduced binding activities to FcγRIIIa F and FcγRIIb ascompared to GpH7-A5/GpH7-B3/GpL16-k0. These results demonstrate thatL234E is an alteration that is effective to increase the bindingactivities to FcγRIIIa F and FcγRIIb when introduced into only one Hchain, while it reduces the binding activities to FcγRIIIa F and FcγRIIbwhen introduced into both H chains.

Then, GpH7-B17 (SEQ ID NO: 11) resulting from the introduction of themutation L234D into GpH7-B3 was constructed according to the methoddescribed in Reference Example 1. Using GGpL16-k0 as the L chain, andGpH7-A5 and GpH7-B17 as the H chain, the heterodimerized antibodyGpH7-A5/GpH7-B17/GpL16-k0 (SEQ ID NOs: 3, 11, and 5, respectively) whosetwo H chains were introduced with L234D was expressed according to themethod described in Reference Example 1. Likewise, using GpH7-B17 as theH chain and GpL16-k0 as the L chain, the homodimerized antibodyGpH7-B17/GpL16-k0 (SEQ ID NOs: 11 and 5, respectively) whose two Hchains were introduced with L234D was expressed according to the methoddescribed in Reference Example 1. These antibodies were assessed for thebinding activity to each FcγR according to the method described inReference Example 2 (FIG. 11). The heterodimerized antibodyGpH7-A5/GpH7-B17/GpL16-k0 had increased binding activity to FcγRIIa Rand FcγRIIb as compared to GpH7-A5/GpH7-B3/GpL16-k0. Meanwhile, thehomodimerized antibody GpH7-B17/GpL16-k0 whose two H chains wereintroduced with the same alteration had reduced binding activities toFcγRIIa R and FcγRIIb as compared to GpH7-A5/GpH7-B3/GpL16-k0. Theseresults demonstrate that L234D is an alteration that is effective toincrease the binding activities to FcγRIIa R and FcγRIIb when introducedinto only one H chain, while it reduces the binding activities toFcγRIIa R and FcγRIIb when introduced into both H chains.

The above findings demonstrate that, even if a homodimerized antibodywhose two H chains have been introduced with the same alterationexhibits reduced FcγR-binding activity, it is possible to increase theFcγR-binding activity by constructing a heterodimerized antibody inwhich only one of the H chains has been introduced with the alteration.

Thus, the above findings demonstrate that a superior FcγR-bindingproperty of an antibody Fc domain can be provided by using aheterodimerized antibody whose two H chains have been introduced withdifferent alterations, as compared to a homodimerized antibody made by aconventional method for introducing the same alteration into both Hchains.

[Example 3] Confirmation of the Direction of FcγR Recognition ofHeterodimerized Antibodies

As shown in Example 2, it was demonstrated that, using heterodimerizedantibodies, higher FcγR-binding activity compared to homodimerizedantibodies can be achieved. When the alterations described in Example 2were introduced into both H chains of an antibody, the FcγR-bindingactivity was rather reduced as compared to the naturally-occurringantibody. This finding suggests that such alteration results in anincrease in the FcγR-binding activity when introduced into one H chainof a heterodimerized antibody; however, when the mutation is introducedinto both H chains, upon binding to FcγR, the residue after alterationincreases the binding in one chain, while inhibits the interaction withFcγR in the other chain. Herein, the state where FcγR is bound in thedirection from the depth side of FIG. 3 is defined as “X-directionbinding”, while in an opposite manner, the binding from the front sideis defined as “Y-direction binding”. It was speculated that in theheterodimerized antibody only either of the X-direction and Y-directionof FcγR-binding activities is altered, whereas in the homodimerizedantibody the FcγR-binding activities in X direction and Y direction arealtered in the same manner.

To prove the hypothesis experimentally, the present inventors foundalterations that mainly inhibit FcγR binding in only either of theX-direction and Y-direction, and introduced such alteration into eitherone of the H chains, and introduced into the same or the other H chainan alteration that enhances the FcγR-binding activity, to verify theinhibition of the FcγR-binding activity. The present inventors aimed tofind a method to be used in combination with the above alteration.Conformational information was searched for alterations that areinvolved in the FcγR binding of an antibody but are only involved in thebinding in only either one of the H chains. As a result, P329 was foundas a candidate. P329 in the H_(A) chain forms a hydrophobic core withTrp at positions 87 and 110 in FcγRIII, while P329 in the H_(B) chaindoes not directly interact with FcγRIII (Nature, 372: 379-383, 1994)(FIG. 12). For example, it was thought that the substitution of P329 ofthe H_(A) chain by an electrically charged residue causes collapse ofthe hydrophobic core, and results in inhibition of the X-directionbinding shown in FIG. 12; however, it was predicted that there is nosignificant impact on the Y-direction binding, because P329 of the H_(B)chain, which is not involved in the Y-direction binding on the otherside, remains unsubstituted.

Mutations, electrically charged R, K, D, and E, were introduced intoGpH7-B3 at P329 to respectively produce the sequences GpH7-B12,GpH7-B13, GpH7-B14, and GpH7-B15 (SEQ ID NOs: 12 to 15), and expressionvectors inserted with these sequences were constructed by the methoddescribed in Reference Example 1. The constructed expression vectorswere each combined with GpH7-A5 and GpL16-k0 to express heterodimerizedantibodies, or they were combined with GpL16-k0 alone but not with otherH chains to express homodimerized antibodies. These antibodies wereexpressed and purified according to the method described in ReferenceExample 1. The resulting purified antibodies are the following: theheterodimerized antibodies GpH7-A5/GpH7-B12/GpL16-k0,GpH7-A5/GpH7-B13/GpL16-k0, GpH7-A5/GpH7-B14/GpL16-k0, andGpH7-A5/GpH7-B15/GpL16-k0; and homodimerized antibodiesGpH7-B12/GpL16-k0, GpH7-B13/GpL16-k0, GpH7-B14/GpL16-k0, andGpH7-B15/GpL16-k0. The prepared antibodies were used to assess thebinding activity to each FcγR by the method described in ReferenceExample 2. The result is shown in FIG. 13.

The FcγR-binding activity of GpH7-A5/GpH7-B12/GpL16-k0 andGpH7-A5/GpH7-B13/GpL16-k0, which are heterodimerized antibodiesintroduced with P329R and P329K, respectively, was about ⅕ to ¼ of thebinding activity to each FcγR of GpH7-A5/GpH7-B3/GpL16-k0. Meanwhile,the FcγR binding was not observed for the homodimerized antibodiesGpH7-B12/GpL16-k0 and GpH7-B13/GpL16-k0. On the other hand, as toFcγRIa, the FcγR-binding activity of GpH7-A5/GpH7-B14/GpL16-k0 andGpH7-A5/GpH7-B15/GpL16-k0, which are the heterodimerized antibodiesintroduced with P329D and P329E, respectively, was about ⅕ to ¼ of thebinding activity to each FcγR of GpH7-A5/GpH7-B3/GpL16-k0; as to FcγRother than FcγRIa, they retained 50% or more of the binding activity.The homodimerized antibodies GpH7-B14/GpL16-k0 and GpH7-B15/GpL16-k0only retained the FcγRIa-binding activity, and the activity was ⅕ orless the FcγRIa-binding activity of GpH7-A5/GpH7-B3/GpL16-k0. GpH7-B12and GpH7-B13 are introduced with basic residues, whereas GpH7-B14 andGpH7-B15 are introduced with acidic residues. Thus, it was thought thatthe FcγR-binding activity is more strongly inhibited by substitutingP329 with a basic residue such as Arg and Lys. The result showing thatthe FcγR-binding activity was maintained when an alteration wasintroduced into only one H chain, while the binding was almostundetectable when the same alteration was introduced into both H chains,supports the hypothesis that the substitution of a hydrophilic residuefor P329 in one H chain inhibits the binding in one direction althoughthe binding in the other direction is retained.

Then, whether the mutations G237A and L234D that enhance theFcγR-binding activity by heterodimerization, which were found asdescribed in Example 2, enhance the interaction with FcγR exclusively inone direction was assessed by comparing the binding activity to eachFcγR of each variant which was prepared to have the alteration P329R incombination with the above alterations. Expression vectors inserted withGpH7-B12 (SEQ ID NO: 12) resulting from the introduction of the mutationP329R into GpH7-B3 such that P329R is introduced into the same H chainas K439E; GpH7-A48 (SEQ ID NO: 16) resulting from the introduction ofthe mutation P329R into GpH7-A5 such that P329R is introduced into thesame H chain as D356K and H435R; GpH7-A45 (SEQ ID NO: 17) resulting fromthe introduction of the mutations G237A and P329R into GpH7-A5; andGpH7-B41 (SEQ ID NO: 18) resulting from the introduction of themutations L234D and P329R into GpH7-B5, were constructed by the methoddescribed in Reference Example 1. Using these expression vectors and theGpL16-k0 expression vector corresponding to an antibody L chain,antibodies were expressed such that P329R and G237A or L234D areintroduced into only either of the H chains according to the methoddescribed in Reference Example 1 (Table 1).

TABLE 1 MUTATION MUTATION SAMPLE H1 SITE H2 SITE GpH7-A5/GpH7- A5 — — B3— — B3/GpL16-k0 (SEQ ID NO: 3, 4, 5) GpH7-A5/GpH7- A5 — — B12 — P329RB12/GpL16-k0 (SEQ ID NO: 3, 12, 5) GpH7-A48/GpH7- A48 — P329R B3 — —B3/GpL16-k0 (SEQ ID NO: 16, 4, 5) GpH7-A26/GpH7- A26 G237A — B3 — —B3/GpL16-k0 (SEQ ID NO: 6, 4, 5) GpH7-A26/GpH7- A26 G237A — B12 — P329RB12/GpL16-k0 (SEQ ID NO: 6, 12, 5) GpH7-A45/GpH7- A45 G237A P329R B3 — —B3/GpL16-k0 (SEQ ID NO: 17, 4, 5) GpH7-A5/GpH7- A5 — — B17 L234D —B17/GpL16-k0 (SEQ ID NO: 3, 11, 5) GpH7-A5/GpH7- A5 — — B41 L234D P329RB41/GpL16-k0 (SEQ ID NO: 3, 18, 5) GpH7-A48/GpH7- A48 — P329R B17 L234D— B17/GpL16-k0 (SEQ ID NO: 16, 11, 5)

The column “SAMPLE” indicates antibody names; the columns “H1” and “H2”indicate names of H chain constant regions of respective antibodies; andthe column “MUTATION SITE” indicates mutations that are differentcompared to GpH7-A5/GpH7-B3/GpL16-k0 (“-”: when there is no particularmutation). The SEQ ID NOs are also shown for the amino acid sequences ofthe H chain and L chain of each antibody.

The combination of G237A and P329R was assessed by usingGpH7-A5/GpH7-B3/GpL16-k0 as a polypeptide for introduction ofalterations; GpH7-A5/GpH7-B12/GpL16-k0 and GpH7-A48/GpH7-B3/GpL16-k0 asvariants in which one of the H chains has been introduced with P329R;GpH7-A45/GpH7-B3/GpL16-k0 as a variant in which G237A has beenintroduced into the same chain as P329R; and GpH7-A26/GpH7-B12/GpL16-k0as a variant in which G237A has been introduced into the other chainthan P329R. They were compared for the binding activity to each FcγRaccording to the method described in Reference Example 2 (FIG. 14).

When GpH7-A5/GpH7-B12/GpL16-k0 in which one of the H chains has beenintroduced with P329R was compared to GpH7-A48/GpH7-B3/GpL16-k0, nosignificant difference was observed in the pattern of FcγR-binding.Thus, it was thought that P329R has no influence on the FcγR bindingeven when introduced into an H chain introduced with D356K and H435R orwhen introduced into an H chain introduced with K439E.

On the other hand, GpH7-A26/GpH7-B3/GpL16-k0, which is a heterodimerizedantibody in which only one of the H chains has been introduced withG237A, showed enhanced binding to FcγRIIa R and IIb as compared toGpH7-A5/GpH7-B3/GpL16-k0. However, as compared toGpH7-A5/GpH7-B12/GpL16-k0 in which only one of the H chains has beenintroduced with P329R, GpH7-A26/GpH7-B12/GpL16-k0 in which the other Hchain has been introduced with G237A exhibited a reduced FcγR binding,and thus the effect of G237A to enhance the binding to FcγRIIa R and IIbwas not observed. Meanwhile, as compared to GpH7-A48/GpH7-B3/GpL16-k0 inwhich only one of the H chains has been introduced with P329R,GpH7-A45/GpH7-B3/GpL16-k0 in which G237A has been introduced with thesame H chain as P329R showed an enhanced binding to FcγRIIa R and IIb,and thus the effect of G237A to enhance the binding to FcγRIIa R and IIbwas observed. Since the binding between the antibody and FcγR wasstrongly inhibited when G237A was introduced into the chain other thanthat introduced with P329R, the above results show that, when G237Aintroduced into GpH7-A5 is combined with P329R introduced into GpH7-B3,they recognize the binding between the antibody and FcγR in the samedirection.

The alteration L234D was also assessed in the same manner (FIG. 15).GpH7-A5/GpH7-B17/GpL16-k0, which is a heterodimerized antibody in whichonly one of the H chains has been introduced with L234D, showed anenhanced binding to FcγRIIa R and IIb as compared toGpH7-A5/GpH7-B3/GpL16-k0. However, as compared toGpH7-A48/GpH7-B3/GpL16-k0 in which only one of the H chains has beenintroduced with P329R, GpH7-A48/GpH7-B17/GpL16-k0 in which L234D hasbeen introduced into the other H chain exhibited a reduced binding toFcγR other than FcγRIa; and thus the effect of L234D to enhance thebinding to FcγRIIa R and IIb was not observed. Meanwhile, as compared toGpH7-A5/GpH7-B12/GpL16-k0 in which only one of the H chains has beenintroduced with P329R, GpH7-A5/GpH7-B41/GpL16-k0 in which L234D has beenintroduced into the same H chain as P329R showed an enhanced binding toFcγRIIa R and IIb; and thus the effect of L234D to enhance the bindingto FcγRIIa R and IIb was observed. Since the binding between theantibody and FcγR was strongly inhibited when L234D was introduced intothe chain other than that introduced with P329R, the above results showthat, when L234D introduced into GpH7-B3 is combined with P329Rintroduced into GpH7-A5, they recognize the binding between the antibodyand FcγR in the same direction.

That is, both G237A and L234D increase (/reduce) the FcγR-bindingactivity of an antibody in the same direction as P329R.

In this way, the hetero-alteration enhanced the binding to FcγRexclusively in either of the X-direction and Y-direction. This findingdemonstrates that the interaction with FcγR is enhanced in an asymmetricmanner in the heterodimerized antibodies.

[Example 4] Comparison of the FcγR Binding of the HeterodimerizedAntibody with that of the Homodimerized Antibody

As described in Example 2, the present inventors revealed that the FcγRbinding of an antibody via its Fc domain can be enhanced by introducingdifferent alterations to the two H chains of an antibody rather thanintroducing the same alteration to both H chains. Thus, to discoveralterations with such a property, the present inventors carried out theexperiment described below.

To construct GpH7-B3 variants, amino acids thought to be involved in theFcγR binding and amino acids around them in GpH7-B3 (SEQ ID NO: 4)constructed as described in Example 2, specifically, Leu at position 234(EU numbering), Leu at position 235 (EU numbering), Gly at position 236(EU numbering), Gly at position 237 (EU numbering), Pro at position 238(EU numbering), Ser at position 329 (EU numbering), Asp at position 265(EU numbering), Val at position 266 (EU numbering), Ser at position 267(EU numbering), His at position 268 (EU numbering), Glu at position 269(EU numbering), Asp at position 270 (EU numbering), Pro at position 271(EU numbering), Gln at position 295 (EU numbering), Tyr at position 296(EU numbering), Ser at position 298 (EU numbering), Tyr at position 300(EU numbering), Ser at position 324 (EU numbering), Asn at position 325(EU numbering), Lys at position 326 (EU numbering), Ala at position 327(EU numbering), Leu at position 328 (EU numbering), Pro at position 329(EU numbering), Ala at position 330 (EU numbering), Pro at position 331(EU numbering), Ile at position 332 (EU numbering), Glu at position 333(EU numbering), Lys at position 334 (EU numbering), Thr at position 335(EU numbering), Ile at position 336 (EU numbering), and Ser at position337 (EU numbering) were each substituted with 18 types of amino acidsexcluding the original amino acid and cysteine. Each GpH7-B3 variant wasnamed “A_B”, where A represents the information on the amino acid typeindicated by one-letter code and the EU numbering of the alteredresidue, and B indicates the information on the amino acid aftersubstitution. For example, when Gly is substituted for Leu at position234 (EU numbering), the B3_variant is named L234_01G. For descriptivepurposes, in the information on the amino acid after substitution, anumeral specific for the amino acid is added before each one-lettercode. Specifically, the symbols used are the following: 01G for Gly; 02Afor Ala; 03V for Val, 04F for Phe; 05P for Pro; 06M for Met; 07I forIle; 08L for Leu; 09D for Asp; 10E for Glu; 11K for Lys; 12R for Arg;13S for Ser; 14T for Thr; 15Y for Tyr; 16H for His; 18N for Asn; 19Q forGln; and 20W for Trp.

Homodimerized antibodies in which both of the H chains have beenintroduced with a mutation were prepared by the following procedure.Antibodies were expressed using a GpH7-B3 variant as the H chain andGpL16-k0 (SEQ ID NO: 5) as the L chain. The antibodies were preparedaccording to the method described in Reference Example 1. Thehomodimerized antibodies in which both of the H chains have beenintroduced with a mutation thus prepared are referred to as Ho Ab.

Heterodimerized antibodies in which only one of the H chains has beenintroduced with a mutation were prepared by the following procedure.Antibodies were expressed using a GpH7-B3 variant and GpH7-A5 (SEQ IDNO: 3) as the H chain and GpL16-k0 (SEQ ID NO: 5) as the L chain. Theantibodies were prepared according to the method described in ReferenceExample 1. The heterodimerized antibodies in which only one of the Hchains has been introduced with a mutation thus prepared are referred toas He Ab.

As a control homodimerized antibody, GpH7-B3/GpL16-k0 that was preparedusing GpH7-B3 (SEQ ID NO: 4) as the H chain and GpL16-k0 (SEQ ID NO: 5)as the L chain was prepared according to the method described inReference Example 1. This control homodimerized antibody is referred toas HoCon Ab. As assessed in Example 2, the binding activity of HoCon Abto each FcγR is not significantly altered as compared to the nativeIgG1.

As a control heterodimerized antibody, GpH7-A5/GpH7-B3/GpL16-k0 that wasprepared using GpH7-A5 (SEQ ID NO: 3) and GpH7-B3 (SEQ ID NO: 4) as theH chain and

GpL16-k0 (SEQ ID NO: 5) as the L chain was prepared according to themethod described in Reference Example 1. This control heterodimerizedantibody is referred to as HeCon Ab. As assessed in Example 2, thebinding activity of HeCon Ab to each FcγR was not significantly alteredas compared to the native IgG1.

Prepared Ho Ab, He Ab, HeCon Ab, and HoCon Ab were assayed for thebinding activity to FcγRIa, FcγRIIa(R), FcγRIIa(H), FcγRIIb, andFcγRIIIa according to the method described in Reference Example 2. As tothe assay result for each FcγR, a graph was drawn by the followingprocedure. The binding activity of He Ab to each FcγR was divided bythat of HeCon Ab, and then multiplied by 100; this is referred to asHe/Con. The binding activity of Ho Ab to each FcγR is divided by that ofHoCon Ab, and then multiplied by 100; this is referred to as Ho/Con.Regarding homodimerized and heterodimerized antibodies prepared using aGpH7-B3 variant comprising an alteration of interest, Ho/Con and He/Convalues were plotted on the horizontal and vertical axes, respectively.The results for respective FcγRs, i.e., FcγRIa, FcγRIIa(R), FcγRIIa(H),FcγRIIb, and FcγRIIIa, are summarized in FIGS. 16 to 20. Based on theHe/Con and Ho/Con values, each alteration can be interpreted as follows.

-   1. When the He/Con and Ho/Con values are 100: it means that the    FcγR-binding activity of the heterodimerized antibody He Ab and    homodimerized antibody Ho Ab, which comprise a GpH7-B3 variant    introduced with a mutation of interest, are equivalent to the    FcγR-binding activity of the control heterodimerized antibody and    control homodimerized antibody, respectively.-   2. When the He/Con and Ho/Con values are 100 or less: it means that    the FcγR-binding activity of the heterodimerized antibody He Ab and    homodimerized antibody Ho Ab, which comprise a GpH7-B3 variant    introduced with a mutation of interest, are weaker than the    FcγR-binding activity of the control heterodimerized antibody and    control homodimerized antibody, respectively.-   3. When the He/Con and Ho/Con values are 100 or more: it means that    the FcγR-binding activity of the heterodimerized antibody He Ab and    homodimerized antibody Ho Ab, which comprise a GpH7-B3 variant    introduced with a mutation of interest, are stronger than the    FcγR-binding activity of the control heterodimerized antibody and    control homodimerized antibody, respectively.-   4. When the He/Con value is greater than the Ho/Con value: it means    that the FcγR-binding activity of the heterodimerized antibody He Ab    comprising a GpH7-B3 variant introduced with a mutation of interest    is stronger than the activity of the homodimerized antibody Ho Ab.-   5. When the He/Con value is smaller than the Ho/Con value: it means    that the FcγR-binding activity of the heterodimerized antibody He Ab    comprising a GpH7-B3 variant introduced with a mutation of interest    is weaker than the activity of the homodimerized antibody Ho Ab.

Based on the interpretation of items 1 to 5 above, the data points inFIGS. 16 to 20 can be classified as shown in FIG. 21.

When an alteration is present in Region i in FIG. 21, it means that thealteration, when introduced into both H chains of a homodimerizedantibody, reduces its binding to FcγR, whereas the same alteration, whenintroduced into only one H chain of a heterodimerized antibody, has theeffect of enhancing its binding to FcγR. That is, the alterationenhances the FcγR binding of the heterodimerized antibody only. As toeach FcγR, alterations comprised in Region i are summarized in Table 2(Tables 2-1 to 2-3), Table 3 (Tables 3-1 and 3-2), Table 4, Table 5, andTable 6.

When an alteration is present in Region ii in FIG. 21, it means that thealteration corresponding the point enhancing the FcγR binding, whenintroduced into both of H chains of a homodimerized antibody, or whenintroduced into only one H chain of a heterodimerized antibody, and theeffect to enhance the binding is stronger in the heterodimerizedantibody. Specifically, alterations in this region have a greater effectof enhancing the FcγR binding in the heterodimerized antibody than inthe homodimerized antibody. As to each FcγR, alterations comprised inRegion ii are summarized in Table 2 (Tables 2-1 to 2-3), Table 3 (Tables3-1 and 3-2), Table 4, Table 5, and Table 6.

In each table, He/Con related to the binding activity to FcγRIa, FcγRIIaH, FcγRIIa R, FcγRIIb, and FcγRIIIa is referred to as He/Con_1a,He/Con_2aH, He/Con_2aR, He/Con_2b, and He/Con_3a, respectively; Ho/Conrelated to the binding activity to FcγRIa, FcγRIIa H, FcγRIIa R,FcγRIIb, and FcγRIIIa is referred to as Ho/Con_1 a, Ho/Con_2aH,Ho/Con_2aR, Ho/Con_2b, and Ho/Con_3a, respectively.

When an alteration is present in Region iii in FIG. 21, it means thatthe alteration corresponding to the point, when introduced into both Hchains of a homodimerized antibody, or when introduced into only one Hchain of a heterodimerized antibody, enhances the FcγR binding, and thebinding enhancement effect is stronger in the homodimerized antibody.Specifically, alterations in this region have a greater effect ofenhancing the FcγR binding in the homodimerized antibody than in theheterodimerized antibody.

In the tables below, amino acid alterations are indicated in a way suchas A327_03V. The first alphabetical letter (for example, “A” ofA327_03V) is a one-letter code representing the amino acid residuebefore alteration, and the following numeral (for example, “327” ofA327_03V) indicates the position of alteration (EU numbering). The lastnumeral and an alphabetical letter (for example, “03V” of A327_03V)indicate an alphabetical letter where the amino acid residue afteralteration is shown by a one-letter code (numeral representing the aminoacid type+ alphabetical letter). Such are indicated as follows:01G(Gly), 02A(Ala), 03V(Val), 04F(Phe), 05P(Pro), 06M(Met), 07I(Ile),08L(Leu), 09D(Asp), 10E(Glu), 11K(Lys), 12R(Arg), 13S(Ser), 14T(Thr),15Y(Tyr), 16H(His), 17C(Cys), 18N(Asn), 19Q(Gln), and 20W(Trp). Thus,for example, “A327_03V” means “a substitution of V for A at amino acidposition 327 (EU numbering)”.

TABLE 2-1 Ia NAME Ho/Con_1a He/Con_1a REGION i A327_03V 94.4 103.2A327_06M 93.1 103.2 A327_07I 95.5 103.5 A327_10E 98.8 103.4 A327_13S86.1 102.4 A330_05P 84.4 101.4 A330_11K 94.1 103.4 E269_01G 97.4 104.4E269_02A 96.2 104.4 E269_03V 95.4 102.6 E269_05P 95.7 102.3 E269_07I97.1 102.8 E269_13S 95.9 103.1 E269_14T 96.2 105.9 E269_16H 98.4 105.8E269_18N 92.7 101.5 E269_19Q 99.6 105.0 E269_20W 98.9 105.2 E333_02A96.2 103.2 E333_03V 98.4 105.0 E333_04F 98.4 102.4 E333_05P 99.2 102.3E333_06M 99.5 102.1 E333_07I 99.9 102.0 E333_08L 99.0 102.3 E333_11K95.8 102.1 E333_12R 94.2 102.2 E333_14T 98.7 102.1 E333_15Y 100.0 101.2E333_16H 99.1 100.8 G236_02A 92.4 100.2 I332_12R 93.2 100.1 L234_03V98.2 104.4 L234_04F 93.5 101.0 L234_06M 96.5 103.8 L234_07I 99.5 103.0L234_10E 79.6 100.5 L234_15Y 98.9 100.1 L328_03V 99.2 101.0 L328_14T98.6 100.4 L328_19Q 99.3 101.1 L328_20W 99.9 102.8 P238_03V 97.9 100.4P238_04F 98.7 101.0 P238_06M 100.0 102.3 P238_08L 100.0 100.9 P238_10E91.8 101.5 P238_15Y 99.0 100.9 P271_14T 99.5 102.3 P271_15Y 99.4 103.0P271_16H 99.4 102.1 P329_01G 89.5 100.9 P329_02A 98.2 102.3 Q295_09D96.8 100.1 Q295_11K 99.9 101.3 Q295_15Y 97.0 101.7 Q295_16H 99.1 102.5REGION ii A327_09D 101.6 102.8 A330_08L 106.1 106.9 A330_09D 108.0 108.6A330_14T 104.8 105.1 A330_16H 103.6 105.8 E269_04F 102.4 103.1 E269_06M101.3 103.6 E269_08L 101.1 103.6 E269_09D 106.3 106.6 E269_15Y 100.2106.4 E333_01G 100.4 102.7 E333_09D 103.2 103.5 E333_13S 100.6 103.1G236_20W 104.4 105.2 H268_02A 105.7 108.7 H268_05P 107.6 109.0 H268_07I106.4 107.8 H268_08L 105.1 107.4 H268_12R 105.3 106.1 H268_14T 109.0112.2 H268_20W 106.2 106.9 I336_01G 104.3 108.7 I336_02A 103.1 106.0I336_03V 104.8 105.2 I336_04F 103.9 105.5 I336_06M 104.0 108.2 I336_10E107.2 107.8 I336_18N 103.6 107.8 K326_12R 100.1 100.1 K326_14T 102.9104.1 K326_15Y 101.2 105.9 K326_16H 100.9 103.4 K326_18N 101.9 105.1K334_06M 104.4 104.6 K334_10E 106.9 109.8 K334_12R 101.7 103.7 K334_20W104.4 105.6 L328_10E 100.6 101.3 P271_02A 102.5 103.5 P271_03V 102.0102.3 P271_04F 100.3 101.8 P271_06M 102.0 102.5 P271_07I 101.9 102.5P271_11K 101.1 102.8 P271_12R 101.2 102.4 P271_13S 100.7 102.4 P271_20W101.5 102.6 Q295_02A 104.1 104.3 Q295_03V 101.6 104.1 Q295_04F 100.3102.2 Q295_05P 101.1 103.4 Q295_07I 101.2 103.0 Q295_08L 103.3 104.6Q295_13S 101.4 102.0 Q295_14T 102.5 102.7 Q295_18N 100.8 102.4 S239_01G100.7 101.3

TABLE 2-2 NAME Ho/Con_1a He/Con_1a REGION i S239_18N 97.9 101.2 S239_19Q93.4 100.6 S267_08L 99.9 101.4 S267_16H 98.2 100.0 S298_11K 90.4 109.3S298_12R 94.5 109.4 S298_20W 71.5 104.1 V266_19Q 91.2 100.2 Y300_12R96.5 104.6 REGION ii S239_09D 100.8 101.0 S239_14T 100.9 101.1 S267_01G102.2 103.2 S267_02A 108.5 108.9 S267_03V 102.4 106.0 S267_09D 105.8109.7 S298_01G 102.8 103.7 S298_02A 106.5 106.6 S298_03V 106.2 109.7S298_04F 106.7 109.0 S298_06M 106.3 114.5 S298_07I 103.3 107.0 S298_08L104.2 106.7 S298_10E 106.1 108.2 S298_14T 106.5 107.1 S298_15Y 103.1107.2 S298_16H 106.8 108.8 S298_18N 103.9 108.1 S298_19Q 104.7 109.4S324_02A 102.6 102.7 S324_03V 103.0 103.6 S324_08L 102.7 103.2 S324_11K100.1 102.1 S324_12R 100.7 101.7 S324_14T 112.4 120.9 S324_15Y 111.7113.8 S324_16H 110.6 112.6 S324_18N 110.8 115.5 S324_19Q 110.7 112.3S324_20W 111.5 114.3 S337_01G 102.3 106.6 S337_02A 103.1 105.8 S337_03V105.1 106.7 S337_04F 104.7 106.2 S337_06M 102.1 106.9 S337_07I 105.3105.3 S337_08L 105.3 105.3 S337_09D 105.5 105.7 S337_10E 104.2 106.4S337_12R 105.4 105.8 S337_15Y 103.6 103.8 S337_19Q 104.6 104.6 S337_20W104.3 104.9 T335_04F 104.8 105.0 T335_05P 106.2 106.4 T335_06M 106.0106.7 T335_07I 105.1 109.1 T335_08L 105.4 114.6 T335_09D 106.8 107.5T335_10E 107.8 108.8 T335_11K 102.8 110.8 T335_12R 106.0 112.8 T335_15Y106.3 107.2

TABLE 2-3 REGION ii NAME Ho/Con_1a He/Con_1a T335_16H 104.5 105.7T335_18N 102.2 105.8 T335_19Q 104.6 105.0 T335_20W 104.2 106.2 Y296_06M103.8 104.0 Y296_10E 105.7 105.8 Y296_11K 102.0 104.7 Y296_12R 102.9106.3 Y296_13S 104.6 105.1 Y296_14T 104.8 105.1 Y296_16H 105.3 105.8Y296_18N 105.0 105.6 Y296_19Q 104.3 111.6 Y296_20W 106.2 110.8 Y300_01G109.0 116.1 Y300_02A 109.8 112.2 Y300_03V 112.4 117.0 Y300_06M 111.0112.2 Y300_08L 109.3 113.1 Y300_09D 111.0 112.5 Y300_10E 110.6 114.4Y300_11K 101.2 106.9 Y300_13S 110.3 111.1 Y300_14T 109.6 111.7 Y300_19Q110.1 112.5 Y300_20W 110.5 112.4

With respect to FcγRIa, alterations comprised in Regions i and ii (FIG.21) are shown.

TABLE 3-1 IIa_R NAME Ho/Con_2aR He/Con_2aR REGION i A330_05P 36.2 114.8A330_15Y 87.5 101.9 A330_19Q 99.1 104.1 E333_04F 92.4 102.1 E333_05P99.9 104.8 E333_07I 98.3 104.4 E333_08L 97.1 103.0 E333_09D 96.5 103.1E333_15Y 96.2 100.7 G236_14T 80.0 102.7 G237_02A 84.0 126.4 G237_06M55.3 133.0 G237_08L 56.6 133.4 G237_09D 73.1 155.6 G237_10E 22.5 116.5G237_13S 44.4 120.7 G237_15Y 76.0 128.6 G237_18N 73.6 150.1 G237_19Q14.3 120.6 H268_05P 95.9 105.6 H268_11K 93.4 100.4 H268_12R 98.5 104.7I332_14T 99.6 105.0 I336_06M 96.4 106.5 I336_18N 94.5 104.1 K326_12R95.7 102.9 K334_20W 96.4 104.8 L234_04F 43.3 104.0 L234_09D 42.8 122.5L234_10E 39.9 112.2 L234_20W 53.8 107.8 L235_04F 98.9 113.6 L235_09D73.0 100.7 L328_02A 97.0 116.7 L328_09D 68.8 119.0 L328_10E 48.1 105.4N325_01G 26.7 106.8 N325_03V 37.8 108.5 N325_04F 65.7 120.3 N325_07I77.0 133.2 N325_08L 95.0 143.8 N325_09D 74.8 116.0 N325_10E 49.9 104.7N325_14T 60.2 105.4 N325_15Y 42.6 100.7 N325_20W 56.0 133.8 P238_06M88.9 139.3 P238_09D 13.0 148.2 P238_10E 21.4 151.3 P238_15Y 95.9 156.2P238_16H 25.5 121.5 P238_19Q 19.4 103.9 P238_20W 25.7 117.4 P271_09D92.4 109.7 P271_10E 88.8 102.6 P331_03V 89.1 114.4 REGION ii A327_09D120.0 123.8 A327_10E 103.7 110.2 A330_01G 105.9 108.9 A330_11K 105.7110.1 A330_12R 100.8 103.5 E269_09D 103.2 105.3 G237_04F 112.8 126.2G237_20W 107.1 138.3 H268_01G 122.0 124.2 H268_03V 104.9 108.6 H268_15Y106.3 109.6 H268_20W 110.5 113.0 I332_06M 105.4 108.8 I336_07I 105.8108.3 I336_08L 105.4 110.2 K326_03V 138.9 140.7 K326_04F 132.7 135.1K326_07I 133.2 147.0 K326_08L 130.7 136.8 K326_15Y 133.8 136.4 K326_18N113.2 115.4 K326_20W 119.9 130.8 K334_02A 100.1 104.9 K334_04F 109.5110.4 K334_05P 101.5 103.1 K334_08L 105.1 106.2 K334_10E 105.7 105.7K334_12R 110.2 110.6 K334_13S 105.6 109.1 K334_19Q 108.1 109.1 L235_15Y110.8 129.3 L235_20W 114.4 130.9 L328_13S 107.9 116.6 L328_14T 118.2118.9 N325_06M 123.7 152.3 N325_13S 125.8 143.4 P238_03V 115.8 120.4P238_04F 146.3 174.4 P271_08L 102.5 103.8 P331_02A 102.2 106.0 P331_04F106.3 110.6 P331_15Y 105.2 112.4 P331_16H 100.2 114.0 P331_20W 101.9109.8 S239_08L 105.6 121.5 S239_10E 121.7 134.9 S239_18N 103.0 110.7S267_03V 114.7 116.5 S267_06M 102.3 122.5 S267_09D 167.1 173.0 S267_19Q110.3 135.2 S298_06M 102.5 108.4 S298_08L 101.4 104.9 S324_04F 100.6104.0 S324_06M 117.9 123.1 S337_02A 101.5 105.0

TABLE 3-2 NAME Ho/Con_2aR He/Con_2aR REGION i P331_06M 79.8 105.9P331_07I 67.0 109.9 P331_08L 74.4 101.7 P331_09D 88.8 102.2 P331_10E94.6 113.3 P331_13S 87.8 104.7 P331_14T 80.8 104.3 P331_18N 76.9 104.6Q295_06M 93.9 101.7 Q295_10E 99.2 101.6 S239_05P 51.2 105.7 S239_06M82.9 100.6 S239_07I 85.3 101.9 S239_14T 92.3 103.4 S337_04F 98.3 104.6S337_08L 99.4 107.4 S337_11K 94.6 102.9 T335_01G 97.7 103.8 T335_12R94.0 100.2 Y296_06M 84.0 101.2 Y296_07I 83.1 101.0 Y296_09D 94.5 105.7Y296_10E 90.2 102.6 Y296_13S 88.1 101.9 Y296_14T 90.3 103.1 Y296_16H89.5 104.7 Y296_18N 93.7 104.1 Y296_19Q 88.9 102.3 Y300_18N 87.6 100.4REGION ii S337_03V 102.0 106.1 S337_06M 100.9 105.2 S337_07I 102.8 108.9S337_09D 113.5 114.8 S337_10E 110.0 111.6 S337_12R 100.4 107.2 S337_15Y100.4 105.4 S337_16H 106.6 108.6 S337_18N 103.1 106.6 S337_19Q 101.8105.6 S337_20W 104.7 110.5 T335_02A 102.3 102.4 T335_05P 103.6 103.6T335_07I 106.7 109.2 T335_10E 108.7 111.3 T335_13S 101.6 109.6 T335_14T106.0 109.8 T335_15Y 107.1 111.4 T335_16H 102.5 108.2 T335_18N 100.4106.3 T335_19Q 102.1 106.0 T335_20W 101.2 108.7 V266_06M 145.6 149.9Y296_04F 106.4 109.4 Y296_20W 106.9 109.8

With respect to FcγRIIa R, alterations comprised in Regions i and ii(FIG. 21) are shown.

TABLE 4 IIa H NAME Ho/Con_2aH He/Con_2aH REGION i A327_09D 90.1 104.7A330_19Q 99.2 102.2 E333_04F 93.6 101.6 E333_08L 96.5 101.5 E333_13S94.6 100.6 E333_14T 90.4 100.4 G236_04F 63.2 117.8 G236_14T 94.2 103.2G236_15Y 80.4 136.1 I336_03V 90.4 102.2 I336_10E 93.4 101.5 I336_18N94.3 103.0 K326_01G 98.2 100.5 K326_07I 78.1 100.2 K326_12R 98.8 100.6K334_13S 94.9 101.6 K334_19Q 97.2 101.3 L234_04F 54.2 119.8 L234_15Y43.8 120.2 L234_20W 55.3 113.5 S239_08L 87.0 104.8 S239_10E 91.4 112.0S267_09D 91.5 105.4 S267_10E 98.5 102.3 S337_07I 94.0 100.8 S337_08L93.1 100.3 S337_20W 96.6 101.9 T335_18N 98.0 103.2 T335_20W 98.7 104.8V266_08L 91.5 104.5 Y296_04F 95.0 100.1 Y296_09D 99.9 104.1 Y296_16H85.7 103.7 REGION ii A330_16H 100.6 101.4 E333_09D 102.7 103.4 G236_03V110.7 119.4 G236_07I 101.0 115.2 H268_02A 110.9 113.8 H268_18N 110.1112.1 H268_19Q 102.3 102.4 I336_06M 100.3 106.1 I336_07I 103.6 104.8I336_08L 100.9 105.5 K326_06M 101.1 103.3 K326_10E 101.0 101.8 K326_19Q100.5 101.4 K334_04F 103.3 105.7 K334_08L 103.4 104.2 K334_16H 101.4103.9 L235_04F 100.7 110.6 L235_15Y 107.1 121.5 L235_20W 111.8 117.7L328_06M 100.2 103.5 L328_14T 110.3 116.2 L328_20W 111.8 112.6 S239_09D112.4 114.4 S267_02A 119.5 119.8 S324_02A 101.5 102.5 S337_09D 106.9107.1 S337_10E 101.3 102.4 T335_01G 100.9 103.1 T335_07I 105.4 107.7T335_10E 105.2 107.6 T335_13S 101.4 106.1 T335_14T 104.3 107.9 T335_15Y101.5 106.3 T335_16H 100.4 105.3 T335_19Q 102.8 103.1 V266_07I 104.3105.5 Y296_18N 100.1 103.8

With respect to FcγRIIa H, alterations comprised in Regions i and ii(FIG. 21) are shown.

TABLE 5 IIb NAME Ho/Con_2b He/Con_2b REGION i A327_07I 37.6 126.7A330_05P 35.3 136.7 G237_02A 47.8 123.6 G237_06M 35.3 129.9 G237_08L49.4 148.0 G237_09D 92.9 222.7 G237_10E 17.8 114.1 G237_13S 25.7 110.8G237_15Y 83.1 152.5 G237_18N 63.9 180.2 G237_19Q 5.3 107.9 I332_02A 81.8103.3 I336_06M 90.1 101.4 L234_09D 68.5 150.4 L234_10E 54.9 117.6L234_20W 80.6 114.4 L235_09D 78.7 106.2 N325_01G 20.2 100.7 N325_03V31.8 111.1 N325_04F 65.7 143.2 N325_07I 88.2 171.4 N325_09D 77.0 124.3N325_20W 44.0 168.8 P238_03V 96.6 120.1 P238_06M 97.1 174.0 P238_10E97.9 232.0 P238_16H 18.8 115.2 P238_19Q 42.7 110.1 P238_20W 12.6 122.7P271_10E 95.0 109.3 P331_03V 86.6 118.7 P331_06M 71.5 101.7 P331_07I63.3 114.5 P331_10E 92.3 115.5 S239_05P 61.0 101.5 S239_06M 84.3 100.9S267_01G 97.0 104.6 S337_07I 96.6 104.0 S337_08L 90.7 100.2 S337_15Y93.4 100.2 S337_18N 98.0 103.3 S337_20W 99.6 108.8 T335_13S 95.2 101.7T335_16H 97.2 102.4 T335_18N 96.1 101.6 T335_19Q 97.3 100.1 T335_20W94.4 103.9 REGION ii A327_18N 100.9 113.1 A330_01G 102.1 103.5 G237_04F139.8 149.2 G237_20W 165.7 193.4 H268_03V 109.1 113.9 H268_05P 109.0116.1 H268_15Y 103.8 106.3 I332_14T 105.8 106.4 I336_07I 100.6 103.4I336_08L 103.0 107.4 K326_07I 169.6 198.8 K326_08L 168.5 176.6 K326_20W131.8 148.5 L235_04F 110.8 118.5 L235_15Y 126.9 149.1 L235_20W 116.3147.3 L328_02A 116.7 144.5 L328_09D 158.7 198.8 L328_10E 146.3 172.2L328_13S 146.6 149.2 N325_06M 159.3 217.0 N325_08L 105.8 196.7 N325_13S159.3 177.2 P238_04F 209.1 312.0 P238_09D 206.6 220.9 P238_15Y 114.2217.8 P271_09D 100.1 120.1 P331_15Y 110.7 114.4 P331_16H 100.4 116.0P331_20W 110.9 111.2 S239_03V 108.0 108.4 S239_10E 175.1 180.6 S267_06M186.7 209.5 S267_19Q 184.9 217.5 S298_06M 101.4 101.8 S324_06M 103.6112.1 S337_16H 102.7 104.6 T335_07I 104.3 106.1 T335_10E 107.7 108.4T335_14T 101.9 106.6 T335_15Y 103.9 108.2 V266_06M 231.3 251.2 Y296_20W102.6 103.3

With respect to FcγRIIb, alterations comprised in Regions i and ii (FIG.21) are shown.

TABLE 6 IIIa NAME Ho/Con_3a He/Con_3a REGION i A330_05P 56.6 155.1A330_07I 93.0 103.1 E333_02A 93.3 105.4 E333_03V 86.5 103.0 E333_04F87.1 101.1 E333_13S 95.1 102.7 E333_20W 90.9 100.9 G236_15Y 41.6 120.8G236_20W 58.3 135.4 H268_18N 90.3 104.1 I332_06M 95.4 101.2 I336_08L95.8 106.3 L234_04F 49.9 118.1 L234_07I 98.2 103.1 L234_09D 52.8 116.8L234_10E 64.7 114.5 L234_15Y 64.7 130.2 L234_20W 37.1 106.8 P238_09D−0.1 105.8 P238_10E 0.8 104.7 P271_01G 88.0 113.2 S324_02A 99.8 104.1S337_04F 95.6 101.4 S337_14T 97.2 101.0 S337_15Y 99.0 101.9 S337_20W87.8 100.8 T335_13S 97.6 105.1 T335_15Y 91.5 104.3 T335_16H 99.5 103.9T335_19Q 97.7 102.2 T335_20W 91.8 101.8 V266_08L 90.5 118.0 REGION iiE333_09D 109.1 112.1 H268_02A 118.0 122.2 I332_01G 103.6 110.7 I336_02A103.6 111.0 I336_03V 110.0 112.2 I336_06M 107.0 108.9 I336_07I 100.2103.1 I336_18N 100.6 104.0 K326_07I 133.5 138.6 K326_08L 113.9 114.2K334_01G 103.6 121.4 K334_09D 149.7 159.7 Q295_14T 108.2 114.7 S267_02A145.3 150.4 S267_09D 159.7 180.8 S298_14T 106.7 112.6 S324_15Y 100.1101.4 S324_16H 103.1 103.7 S337_09D 105.5 109.9 S337_10E 100.2 105.0S337_18N 101.5 104.1 T335_07I 107.0 107.2 T335_10E 102.4 106.9 T335_14T102.5 108.5

With respect to FcγRIIIa, alterations comprised in Regions i and ii(FIG. 21) are shown.

The alterations revealed in this Example, together with Example 2, arethought to support the concept of the improvement of FcγR recognitionability by the heterodimerized antibody described in Example 1.Alterations in Region i in FIG. 21 are thought to reduce theFcγR-binding activity when introduced into both H chains in aconventional way, and have not been recognized as alterations thatenhance the binding. The present inventors, however, succeeded indemonstrating that such alterations also improve the FcγR-bindingactivity by introducing them into only one of the two H chains.

[Example 5] Method for Determining Combinations of Alterations ofHeterodimerized Antibodies

As described in Example 3, the effect of an alteration on the FcγRbinding of a heterodimerized antibody differs in the direction dependingon alteration. For this reason, when several alterations are combined tofurther enhance or reduce the FcγR binding of a heterodimerizedantibody, it is necessary to unify the direction of the effect of eachalteration on the FcγR binding. If two alterations were introduced in away that their effects to enhance the FcγR binding of an antibody aredifferent in the direction, the effects of the alterations wouldannihilate each other and the effect of enhancing the binding would notbe observed in spite of the combination of alterations for enhancing theFcγR binding. However, there is no method for predicting in advancewhich H chain should be introduced with an alteration. Thus, in order tofind an appropriate method of combination, generally antibodies in whichalterations of interest are introduced into the same chain or differentH chains need to be prepared, and they are compared to each other forthe FcγR-binding activity. For example, when all of three differenttypes of alterations are introduced into a homodimerized antibody, asingle type of antibody in which each alteration has been introducedinto both H chains may be prepared. In the case of a heterodimerizedantibody, however, it is necessary to determine which H chain should beintroduced with each alteration. In this case, as shown in FIG. 22,there are a maximum of four combinations to be tried; i.e., the numberof variants that should be prepared for assessment of the alterationcombinations is very large, and this is inefficient. Thus, the presentinventors tested a method for identifying the direction in which analteration of interest has an effect on the FcγR binding of an antibodyby combining the alteration with P329R that inhibits the FcγR binding ofan antibody from only one direction.

The present inventors examined how the FcγRIIIa binding was altered whenalterations L234Y, G236W, and S298A that enhance the FcγRIIIa bindingwere each introduced into the same H chain as P329R or the other H chainof a heterodimerized antibody found in Example 4. First, variants ofinterest were expressed and prepared according to the method describedin Reference Example 1, using GpH7-A5 as an H chain; and GpL16-k0 as theL chain; and as the other H chain, GpH7-HA5 (SEQ ID NO: 19), GpH7-HA6(SEQ ID NO: 20), and GpH7-HA 11 (SEQ ID NO: 21) resulting from theintroduction of L234Y, G236W, and S298A, respectively, into GpH7-B12introduced with the alteration P329R. Furthermore, antibodies ofinterest were expressed and prepared according to the method describedin Reference Example 1, using as an H chain, GpH7-A48 (SEQ ID NO: 16)resulting from the introduction of P329R into GpH7-A5; and GpL16-k0 asthe L chain; and as the other H chain, GpH7-B3-01-15Y (SEQ ID NO: 22),GpH7-B3-03-20W (SEQ ID NO: 23), and GpH7-B3-15-02A (SEQ ID NO: 24)resulting from the introduction of L234Y, G236W, and S298A,respectively, into GpH7-B3. The antibodies thus obtained were namedGpH7-A5/GpH7-HA5/GpL16-k0 (SEQ ID NOs: 3, 19, and 5, respectively),GpH7-A5/GpH7-HA6/GpL16-k0 (SEQ ID NOs: 3, 20, and 5, respectively),GpH7-A5/GpH7-HA11/GpL16-k0 (SEQ ID NOs: 3, 21, and 5, respectively),GpH7-A48/GpH7-B3-01-15Y/GpL16-k0 (SEQ ID NOs: 16, 22, and 5,respectively), GpH7-A48/GpH7-B3-03-20W/GpL16-k0 (SEQ ID NOs: 16, 23, and5, respectively), and GpH7-A48/GpH7-B3-15-02A/GpL16-k0 (SEQ ID NO: 16,24, and 5, respectively). The variants were compared to each other forthe FcγRIIIa-binding activity according to the method described inReference Example 2. The effects of the combination of L234Y, G236W, andS298A with P329R are summarized in Table 7.

TABLE 7 FcγRIIIa MUTATION MUTATION BINDING SAMPLE H1 SITE H2 SITEACTIVITY GpH7-A5/GpH7-B3/GpL16-k0 A5 — B3 — — 100 (SEQ ID NO: 3, 4, 5)GpH7-A48/GpH7-B3-01-15Y/GpL16-k0 A48 P329R B3-01-15Y L234Y — 11 (SEQ IDNO: 16, 22, 5) GpH7-A5/GpH7-HA5/GpL16-k0 A5 — HA5 L234Y P329R 60 (SEQ IDNO: 3, 19, 5) GpH7-A48/GpH7-B3-03-20W/GpL16-k0 A48 P329R B3-03-20W G236W— 13 (SEQ ID NO: 16, 23, 5) GpH7-A5/GpH7-HA6/GpL16-k0 A5 — HA6 G236WP329R 56 (SEQ ID NO: 3, 20, 5) GpH7-A48/GpH7-B3-15-02A/GpL16-k0 A48P329R B3-15-02A S298A — 47 (SEQ ID NO: 16, 24, 5)GpH7-A5/GpH7-HA11/GpL16-k0 A5 — HA11 S298A P329R 84 (SEQ ID NO: 3, 21,5)

The column “SAMPLE” indicates antibody names; the columns “H1” and “H2”indicate names of the H chain constant region of respective antibodies;and the column “MUTATION SITE” indicates mutations that are different incomparison with GpH7-A5/GpH7-B3/GpL16-k0 (“-”: when there is noparticular mutation). The FcγRIIIa-binding activity is indicated as arelative binding activity when setting the FcγRIIIa binding ofGpH7-A5/GpH7-B3/GpL16-k0 (SEQ ID NO: 3, 4, and 5, respectively) as 100.The SEQ ID NOs are also shown for the amino acid sequences of the Hchain and L chain of each antibody.

Based on the result, the alterations were compared for theFcγRIIIa-binding activity when each alteration has been introduced intothe same H chain as P329R or the H chain different from that introducedwith P329R. In the case of L234Y, the binding activity ofGpH7-A5/GpH7-HA5/GpL16-k0 corresponding to the former was 60, while thebinding activity of GpH7-A48/GpH7-B3-01-15Y/GpL16-k0 corresponding tothe latter was 11; thus, the FcγR binding was inhibited when thealteration was introduced into the H chain different from thatintroduced with P329R. In the case of G236W, the binding activity ofGpH7-A5/GpH7-HA6/GpL16-k0 corresponding to the former was 56, while thebinding activity of GpH7-A48/GpH7-B3-03-20W/GpL16-k0 corresponding tothe latter was 13; thus, the FcγR binding was inhibited when thealteration was introduced into the H chain different from thatintroduced with P329R. In the case of S298A, the binding activity ofGpH7-A5/GpH7-HA11/GpL16-k0 corresponding to the former was 84, while thebinding activity of GpH7-A48/GpH7-B3-15-02A/GpL16-k0 corresponding tothe latter was 47; thus, the FcγR-binding activity was inhibited whenthe alteration was introduced into the H chain different from thatintroduced with P329R. For all the alterations, the FcγRIIIa binding wasinhibited when an alteration was introduced into the H chain differentfrom that introduced with P329R, as described as the latter case. If theH chain introduced with P329R corresponded to the H_(A) chain of FIG. 3,P329R is thought to inhibit the binding in the X-direction. Sincecombinations that resulted in significant inhibition of the binding arethose when L234Y, G236W, or S298A were introduced into the H chaindifferent from that introduced with P329R, in this case the alterationswould have been introduced into the H_(B) chain. Since the effect toenhance the FcγRIIIa binding was markedly inhibited when any of thealterations L234Y, G236W, and S298A was introduced into the H chaindifferent from that introduced with P329R, all the alterations, whenintroduced into the H_(B) chain, enhanced the FcγRIIIa binding from theX-direction, which is inhibited by P329R. Thus, it was thought that theFcγRIIIa binding can be further enhanced by introducing thesealterations into the same H chain.

The above-described hypothesis was tested by assessing whether the FcγRbinding is enhanced by introducing two of L234Y, G236W, and S298A intothe same H chain or different H chains. Expression vectors inserted withGpH7-TA1 (SEQ ID NO: 25), GpH7-TA2 (SEQ ID NO: 26), and GpH7-TA3 (SEQ IDNO: 27), resulting from the introduction of L234Y, G236W, and S298A intoGpH7-A5, respectively; and expression vectors inserted withGpH7-B3-01-15Y (SEQ ID NO: 22), GpH7-B3-03-20W (SEQ ID NO: 23), andGpH7-B3-15-02A (SEQ ID NO: 24), resulting from the introduction ofL234Y, G236W, and S298A into GpH7-B3, respectively, were constructedaccording to the method described in Reference Example 1. In addition,expression vectors inserted with the following were constructed:GpH7-TA4 (SEQ ID NO: 28) resulting from the introduction of L234Y andG236W into GpH7-A5; GpH7-TA5 (SEQ ID NO: 29) resulting from theintroduction of L234Y and S298A into GpH7-A5; and GpH7-TA6 (SEQ ID NO:30) resulting from the introduction of G236W and S298A into GpH7-A5.These were combined in a manner as shown in Table 8, and GpL16-k0 wasadded as the L chain to each combination. Antibodies of interest wereexpressed and prepared according to the method described in ReferenceExample 1. Regarding the expressed samples, information on the H chainsand mutation sites, and assay results on the FcγRIIIa binding of theantibody are summarized in Table 8.

TABLE 8 FcγRIIIa MUTATION MUTATION BINDING SAMPLE H1 SITE H2 SITEACTIVITY GpH7-A5/GpH7-B3-01-15Y/GpL16-k0 A5 — — B3-01-15Y L234Y 131 (SEQID NO: 3, 22, 5) GpH7-A5/GpH7-B3-03-20W/GpL16-k0 A5 — — B3-03-20W G236W140 (SEQ ID NO: 3, 23, 5) GpH7-A5/GpH7-B3-15-02A/GpL16-k0 A5 — —B3-15-02A S298A 163 (SEQ ID NO: 3, 24, 5)GpH7-TA2/GpH7-B3-01-15Y/GpL16-k0 TA2 — G236W B3-01-15Y L234Y 130 (SEQ IDNO: 26, 22, 5) GpH7-TA4/GpH7-B3/GpL16-k0 TA4 L234Y G236W B3 — 168 (SEQID NO: 28, 4, 5) GpH7-TA1/GpH7-B3-15-02A/GpL16-k0 TA1 L234Y — B3-15-02AS298A 142 (SEQ ID NO: 25, 24, 5) GpH7-TA5/GpH7-B3/GpL16-k0 TA5 L234YS298A B3 — 208 (SEQ ID NO: 29, 4, 5) GpH7-TA3/GpH7-B3-03-20W/GpL16-k0TA3 — S298A B3-03-20W G236W 70 (SEQ ID NO: 27, 23, 5)GpH7-TA6/GpH7-B3/GpL16-k0 TA6 G236W S298A B3 — 228 (SEQ ID NO: 30, 4, 5)

The column “SAMPLE” indicates antibody names; the columns “H1” and “H2”indicate names of the H chain constant region of respective antibodies;and the column “MUTATION SITE” indicates mutations that are different incomparison with GpH7-A5/GpH7-B3/GpL16-k0 (“-”: when there is noparticular mutation). The FcγRIIIa-binding activity is indicated as arelative binding activity when setting the FcγRIIIa binding ofGpH7-A5/GpH7-B3/GpL16-k0 as 100. The SEQ ID NOs are also shown for theamino acid sequences of the H chain and L chain of each antibody.

The effect of the combination of L234Y and G236W was assessed based onthe result shown in Table 8. The FcγRIIIa-binding activity ofGpH7-TA2/GpH7-B3-01-15Y/GpL16-k0 in which L234Y and G236W wereintroduced into different H chains was 130, and was not increased ascompared to the binding activity of 131 ofGpH7-A5/GpH7-B3-01-15Y/GpL16-k0 introduced with L234Y alone, while itwas decreased as compared to the binding activity of 140 ofGpH7-A5/GpH7-B3-03-20W/GpL16-k0 introduced with G236W alone. Meanwhile,the binding activity of GpH7-TA4/GpH7-B3/GpL16-k0 in which L234Y andG236W were introduced into the same H chain was 168, and was increasedas compared to the binding activity of GpH7-A5/GpH7-B3-01-15Y/GpL16-k0introduced with L234Y alone and GpH7-A5/GpH7-B3-03-20W/GpL16-k0introduced with G236W alone. As predicted, this result demonstrates thatL234Y and G236W, when introduced into the same H chain, further increasethe FcγRIIIa-binding activity.

Then, the effect of the combination of L234Y and S298A was assessedbased on Table 8. The FcγRIIIa-binding activity ofGpH7-TA1/GpH7-B3-15-02A/GpL16-k0 in which L234Y and S298A wereintroduced into different H chains was 142, and was increased ascompared to the binding activity of 131 ofGpH7-A5/GpH7-B3-01-15Y/GpL16-k0 introduced with L234Y alone, while itwas decreased as compared to the binding activity of 163 ofGpH7-A5/GpH7-B3-15-02A/GpL16-k0 introduced with S298A alone. That is,since the binding activity of GpH7-TA1/GpH7-B3-15-02A/GpL16-k0 was notincreased as compared to when S298A alone was introduced, it can be saidthat the effect to further increase the FcγRIIIa-binding activity wasnot provided when S298A and L234Y were introduced into different Hchains. Meanwhile, the FcγRIIIa-binding activity ofGpH7-TA5/GpH7-B3/GpL16-k0 in which L234Y and S298A were introduced intothe same H chain was 208, and was increased as compared toGpH7-A5/GpH7-B3-01-15Y/GpL16-k0 introduced with L234Y alone andGpH7-A5/GpH7-B3-15-02A/GpL16-k0 introduced with S298A alone. Aspredicted, this result demonstrates that L234Y and S298A, whenintroduced into the same H chain, further increase the FcγRIIIa-bindingactivity.

Next, the effect of the combination of G236W and S298A was assessedbased on Table 8. The FcγRIIIa-binding activity ofGpH7-TA3/GpH7-B3-03-20W/GpL16-k0 in which G236W and S298A wereintroduced into different H chains was 70, and was decreased as comparedto the binding activity of 140 of GpH7-A5/GpH7-B3-03-20W/GpL16-k0introduced with G236W alone, and the binding activity of 163 ofGpH7-A5/GpH7-B3-15-02A/GpL16-k0 introduced with S298A alone. Meanwhile,the binding activity of GpH7-TA6/GpH7-B3/GpL16-k0 in which G236W andS298A were introduced into the same H chain was 228, and was increasedas compared to GpH7-A5/GpH7-B3-03-20W/GpL16-k0 introduced with G236Walone and GpH7-A5/GpH7-B3-15-02A/GpL16-k0 introduced with S298A alone.As predicted, this result demonstrates that G236W and S298A, whenintroduced into the same H chain, further increase the FcγRIIIa-bindingactivity.

As was initially predicted, these results demonstrate that L234Y, G236W,and S298A, only when each is introduced into the same H chain, enhancethe binding. This data supports that L234Y, G236W, and S298A, when beingpresent in the same chain, enhance the FcγR binding from the samedirection. That is, this shows that one can determine a method forappropriately combining two alterations based on a result predicted fromthe result of comparison of the FcγRIIIa-binding activity, combiningP329R with each of the alterations. In other words, combining with P329Ris a useful method for predicting a method of combining alterations inheterodimerized antibodies. This method can be used to reveal otheruseful combinations of alterations.

Combinations of two or more alterations were considered based on theresult of comparison of the FcγRIIIa-binding activity combining P329Rwith each of the alterations. It was demonstrated that L234Y and G236W,G236W and S298A, and S298A and L234Y, when introduced into the same Hchain, respectively enhance the interaction with FcγRIIIa from the samedirection. That is, from this result, it was thought that L234Y, G236W,and S298A all enhance the FcγRIIIa-binding activity from the samedirection, and thus these alterations, when introduced into the same Hchain, were expected to maximally enhance the FcγRIIIa-binding activity.To assess this hypothesis, GpH7-TA4, GpH7-TA5, and GpH7-TA6 wereconstructed by introducing into GpH7-A5 each of the alteration groups ofL234Y and G236W, L234Y and S298A, and G236W and S298A; GpH7-B3-01-15Y,GpH7-B3-03-20W, and GpH7-B3-15-02A were constructed by introducingL234Y, G236W, and S298A, respectively, into GpH7-B3; expression vectorsinserted with the above constructs were prepared according to ReferenceExample 1. The vectors were combined in a way that the threealterations, L234Y, G236W, and S298A, were introduced into either Hchain; and GpL16-k0 was added as the L chain; and antibodies of interestwere expressed and prepared according to the method described inReference Example 1. Furthermore, GpH7-TA7 (SEQ ID NO: 31) resultingfrom the introduction of three alterations, L234Y, G236W, and S298A intoGpH7-A5 was constructed, and combined with GpH7-B3 and GpL16-k0 toexpress and purify an antibody of interest according to the methoddescribed in Reference Example 1. A list of antibodies prepared asdescribed herein and the result of comparison of the FcγRIIIa-bindingactivity between antibodies are shown in Table 9.

TABLE 9 FcγRIIIa MUTATION BINDING SAMPLE H1 MUTATION SITE H2 SITEACTIVITY GpH7-TA4/GpH7-B3-15-02A/GpL16-k0 TA4 L234Y G236W — B3-15-02AS298A 151 (SEQ ID NO: 28, 24, 5) GpH7-TA5/GpH7-B3-03-20W/GpL16-k0 TA5L234Y S298A — B3-03-20W G236W 97 (SEQ ID NO: 29, 23, 5)GpH7-TA6/GpH7-B3-01-15Y/GpL16-k0 TA6 G236W S298A — B3-01-15Y L234Y 103(SEQ ID NO: 30, 22, 5) GpH7-TA7/GpH7-B3/GpL16-k0 TA7 L234Y G236W S298AB3 — 201 (SEQ ID NO: 31, 4, 5)

The column “SAMPLE” indicates antibody names; the columns “H1” and “H2”indicate names of the H chain constant region of respective antibodies;and the column “MUTATION SITE” indicates mutations that are different incomparison with GpH7-A5/GpH7-B3/GpL16-k0 (“-”: when there is noparticular mutation). The FcγRIIIa-binding activity is indicated as arelative binding activity when setting the FcγRIIIa-binding activity ofGpH7-A5/GpH7-B3/GpL16-k0 as 100. The SEQ ID NOs are also shown for theamino acid sequences of the H chain and L chain of each antibody.

As was predicted from the result of comparison of the FcγRIIIa-bindingactivity combining P329R with each alteration, GpH7-TA7/GpH7-B3/GpL16-k0in which L234Y, G236W, and S298A were introduced into the same H chainexhibited most strongly enhanced FcγRIIIa binding. That is, the resultdemonstrates that one can predict a method for appropriately combiningtwo or more alterations by comparing the FcγRIIIa-binding activitycombining P329R with each alteration.

[Example 6] Comparison of Conventional Homodimerized Antibody and NovelHeterodimerized Antibody Based on Heterodimerized Antibody

The results shown in Tables 7, 8, and 9 of Example 5 demonstrate thathetero-alterations which each alone enhances the FcγRIIIa-bindingactivity, when appropriately combined together, can further enhance theFcγRIIIa binding. Specifically, it was demonstrated that the alterationsL234Y, G236W, and S298A, when introduced into the same H chain, furtherincrease the FcγR-binding activity.

Then, the present inventors examined whether, even multiple alterationsare combined, the resulting heterodimerized antibody still had thecharacteristic of the heterodimerized antibody that the heterodimerizedantibody introduced with multiple alterations exhibits more stronglyenhanced FcγR binding than the homodimerized antibody introduced withthe corresponding multiple alterations. Specifically, GpH7-TA7 andGpH7-TA45 (SEQ ID NO: 32) resulting from the introduction of L234Y,G236W, and S298A into GpH7-A5 and GpH7-B3, respectively, were preparedaccording to the method described in Reference Example 1. As shown inTable 10, the heterodimerized antibodies GpH7-TA7/GpH7-B3/GpL16-k0 andGpH7-A5/GpH7-TA45/GpL16-k0 in which L234Y, G236W, and S298A wereintroduced into only one H chain, and the homodimerized antibodyGpH7-TA7/GpH7-TA45/GpL16-k0 in which L234Y, G236W, and S298A wereintroduced into both H chains were expressed and purified according tothe method described in Reference Example 1. These antibodies werecompared for the FcγRIIIa binding according to the method described inReference Example 2 (Table 10).

TABLE 10 FcγR BINDING SAMPLE H1 MUTATION SITE H2 MUTATION SITE ACTIVITYGpH7-A5/GpH7-B3/GpL16-k0 A5 — — — B3 — — — 100 (SEQ ID NO: 3, 4, 5)GpH7-TA7/GpH7-B3/GpL16-k0 TA7 L234Y G236W S298A B3 — — — 210 (SEQ ID NO:31, 4, 5) GpH7-A5/GpH7-TA45/GpL16-k0 A5 — — — TA45 L234Y G236W S298A 225(SEQ ID NO: 3, 32, 5) GpH7-TA7/GpH7-TA45/GpL16-k0 TA7 L234Y G236W S298ATA45 L235Y G237W S299A 48 (SEQ ID NO: 31, 32, 5)

The column “SAMPLE” indicates antibody names; the columns “H1” and “H2”indicate names of the H chain constant region of respective antibodies;and the column “MUTATION SITE” indicates mutations that are different incomparison with GpH7-A5/GpH7-B3/GpL16-k0 (“-”: when there is nomutation). The FcγRIIIa-binding activity is indicated as a relativebinding activity when setting the FcγRIIIa binding ofGpH7-A5/GpH7-B3/GpL16-k0 as 100. The SEQ ID NOs are also shown for theamino acid sequences of H chain and L chain of each antibody.

The result shown in Table 10 demonstrates that the homodimerizedantibody GpH7-TA7/GpH7-TA45/GpL16-k0 in which both of the H chains wereintroduced with L234Y, G236W, and S298A exhibited reduced FcγRIIIabinding as compared to the heterodimerized antibodiesGpH7-TA7/GpH7-B3/GpL16-k0 and GpH7-A5/GpH7-TA45/GpL16-k0 in which onlyone of the H chains were introduced with L234Y, G236W, and S298A. Thisshows that the characteristic of the alterations L234Y, G236W, and S298Athat they increase the FcγR-binding activity of a heterodimerizedantibody but reduces the FcγR-binding activity of a homodimerizedantibody, is retained even when multiple alterations are combinedtogether.

Then, the present inventors examined whether a heterodimerized antibodyin which only one of the H chains was introduced with L234Y, G236W, andS298A retains the directionality of the FcγRIIIa binding discussed inExample 3.

GpH7-TA8 (SEQ ID NO: 33) and GpH7-B12 (SEQ ID NO: 12) resulting from theintroduction of P329R into GpH7-TA7 and GpH7-B3, respectively, wereprepared according to the method described in Reference Example 1. Asshown in Table 11, the heterodimerized antibodyGpH7-TA8/GpH7-B3/GpL16-k0 in which L234Y, G236W, and S298A wereintroduced into the same H chain as P329R, and the heterodimerizedantibody GpH7-TA7/GpH7-B12/GpL16-k0 in which L234Y, G236W, and S298Awere introduced into the H chain different from that introduced withP329R were prepared according to the method described in ReferenceExample 1. As to the FcγRIII-binding activity, these antibodies werecompared with the heterodimerized antibody GpH7-TA7/GpH7-B3/GpL16-k0 inwhich one of the H chains was introduced with L234Y, G236W, and S298Aaccording to the method described in Reference Example 2 (Table 11).

TABLE 11 FcγRIIIa MUTATION BINDING SAMPLE H1 MUTATION SITE H2 SITEACTIVITY GpH7-A5/GpH7-B3/GpL16-k0 A5 — — — — B3 — 100 (SEQ ID NO: 3, 4,5) GpH7-TA7/GpH7-B3/GpL16-k0 TA7 L234Y G236W S298A — B3 — 210 (SEQ IDNO: 31, 4, 5) GpH7-TA7/GpH7-B12/GpL16-k0 TA7 L234Y G236W S298A — B12P329R 11 (SEQ ID NO: 31, 12, 5) GpH7-TA8/GpH7-B3/GpL16-k0 TA8 L234YG236W S298A P329R B3 — 150 (SEQ ID NO: 33, 4, 5)

The column “SAMPLE” indicates antibody names; the columns “H1” and “H2”indicate names of the H chain constant region of respective antibodies;and the column “MUTATION SITE” indicates mutations that are different incomparison with GpH7-A5/GpH7-B3/GpL16-k0 (“-”: when there is noparticular mutation). The FcγRIIIa-binding activity is indicated as arelative binding activity when setting the FcγRIIIa binding ofGpH7-A5/GpH7-B3/GpL16-k0 as 100. The SEQ ID NOs are also shown for theamino acid sequences of H chain and L chain of each antibody.

The result shown in Table 11 demonstrates that the heterodimerizedantibody GpH7-TA8/GpH7-B3/GpL16-k0 in which the group of alterationsL234Y, G236W, and S298A was introduced into the same H chain as P329R,and the heterodimerized antibody GpH7-TA7/GpH7-B12/GpL16-k0 in which thegroup of alterations L234Y, G236W, and S298A was introduced into the Hchain different from that introduced with P329R both exhibited reducedFcγRIIIa-binding activities as compared to GpH7-TA7/GpH7-B3/GpL16-k0.The FcγRIIIa-binding activity of GpH7-TA7/GpH7-B12/GpL16-k0 was 11, andwas markedly reduced as compared to the activity of 150 ofGpH7-TA8/GpH7-B3/GpL16-k0. This result shows the characteristic that theFcγR-binding activity is markedly reduced if multiple of the alterationsL234Y, G236W, and S298A are introduced into an H chain, when they areintroduced into the H chain different from that introduced with P329R,which was observed in Example 3 for the alterations L234Y, G236W, andS298A.

The above findings demonstrate that an appropriate combination ofalterations that enhance the FcγR binding can further increase theFcγR-binding activity while keeping the characteristic of aheterodimerized antibody.

[Example 7] Comparison with Prior Art: Comparison BetweenHeterodimerized Variants and Amino Acid-Altered Antibodies that EnhanceFcγRIIIa Binding

Alterations that increase the ADCC activity by enhancement of theFcγRIIIa binding are already known. For example, the alterations S239D,I332E, and A330L are known as mutations that enhance the FcγRIIIabinding the most when introduced into both H chains of an antibody(Proc. Natl. Acad. Sci. USA, 103, 4005-4010, 2006). The antitumoractivity of an antibody was demonstrated to be increased by theenhancement of the antibody-dependent cellular cytotoxic (ADCC)activity. Enhancing the FcγRIIIa-binding of an antibody is an effectivemeans to enhance the utility of antibodies as pharmaceuticals. However,as shown in the Examples above, there are thought to be limitations onthe enhancement of the FcγR-binding activity using homodimerizedantibodies. Thus, it was thought that the FcγR-binding activity can befurther increased by hetero-alteration.

As mentioned in Example 1, the Fc domain of an antibody interacts withFcγR in an asymmetric manner. In the case of an antibody introduced withthe alterations S239D, I332E, and A330L, in view of thethree-dimensional structure, it is thought that, in the H_(A) chain, allof the altered residues of S239D, I332E, and A330L are involved in theenhancement of the interaction with FcγR, while in the H_(B) chain,residues other than S239D are out of contact with FcγR and do notcontribute to the enhancement of the FcγR-binding activity (FIG. 23).That is, in light of the asymmetry of interaction between the Fc domainand FcγR, each alteration introduced by the conventional antibodyalteration technology is not sufficiently capable of interaction withFcγR and this is thought to be sufficient for optimizing theantibody/FcγR interaction. For example, in the case of above-describedalterations S239D, I332E, and A330L, it is thought that the FcγRIIIabinding can be further enhanced by introducing alterations that enhancethe interaction with FcγRIIIa on the H_(B) chain side, instead ofintroducing the above alterations into H_(B) chain. That is, the FcγRbinding could be further enhanced by using the technology of the presentinvention for producing heterodimerized antibodies by introducingdifferent alterations to each antibody H chain (hereinafter referred toas “heterodimerized antibody technology”) than using the technology forintroducing the same alteration to both antibody H chain (hereinafterreferred to as “prior art” or “homodimerized antibody technology”).

In view of the three-dimensional structure of the Fc/FcγRIIIa complex,contrary to S239D, I332E, and A330L, it is thought that S298 interactswith FcγR only in the H_(B) chain as shown in FIG. 23 (JBC, 276:16469-16477, 2001). Thus, it is thought that, when an alteration isintroduced at S298, the residue after substitution mutation alsointeracts with FcγRIIIa on the H_(B) chain side. As seen in Example 5,L234Y and G236W are thought to enhance the interaction with FcγR fromthe same direction as S298A. That is, it is thought that, when S239D,A330L, and I332E are introduced into the same H chain, and L234Y, G236W,and S298A are introduced into the other H chain, all of the introducedalterations can interact with FcγR at the same time, resulting infurther enhancement of the interaction with FcγR.

To assess the above hypothesis, the present inventors carried out thefollowing experiments. The direction of FcγR recognition by eachalteration was determined according to the method described in Example5, using antibodies having an H chain introduced with the alterationL234Y, G236W, S298A, S239D, A330L, or I332E, and P329R introduced intothe same or different H chain. Expression vectors inserted with thefollowing were constructed according to the method described inReference Example 1:

GpH7-A5; GpH7-A48 (SEQ ID NO: 16) resulting from the introduction ofP329R into GpH7-A5; GpH7-HA7 (SEQ ID NO: 34) resulting from theintroduction of S239D and P329R into GpH7-B3; GpH7-HA15 (SEQ ID NO: 35)resulting from the introduction of A330L and P329R into GpH7-B3;GpH7-HA18 (SEQ ID NO: 36) resulting from the introduction of I332E andP329R into GpH7-B3; GpH7-HA5 (SEQ ID NO: 19) resulting from theintroduction of L234Y and P329R into GpH7-B3; GpH7-HA6 (SEQ ID NO: 20)resulting from the introduction of G236W and P329R into GpH7-B3;GpH7-HA11 (SEQ ID NO: 21) resulting from the introduction of S298A andP329R into GpH7-B3; GpH7-B3-06-09D (SEQ ID NO: 37) resulting from theintroduction of S239D into GpH7-B3; GpH7-B3-20-08L (SEQ ID NO: 38)resulting from the introduction of A330L into GpH7-B3; GpH7-B3-22-10E(SEQ ID NO: 39) resulting from the introduction of I332E into GpH7-B3;GpH7-B3-01-15Y (SEQ ID NO: 22) resulting from the introduction of L234Yinto GpH7-B3; GpH7-B3-03-20W (SEQ ID NO: 23) resulting from theintroduction of G236W into GpH7-B3; and GpH7-B3-15-02A (SEQ ID NO: 24)resulting from the introduction of S298A into GpH7-B3. The expressionvectors for the respective H chains were combined in a way that each ofthe alterations L234Y, G236W, S298A, S239D, A330L, and I332E is presentin the same H chain as P329R or in the different H chain from P329R, andthe expression vector GpL16-k0 for the L chain was combined with them.Antibodies of interest were expressed and prepared according to themethod described in Reference Example 1. The prepared antibodies wereused to assay the FcγRIIIa-binding activity. The result of assessmentusing P329R on the direction of FcγRIIIa recognition by L234Y, G236W,S298A, S239D, A330L, and I332E is summarized in Table 12.

TABLE 12 FcγRIIIa MUTATION BINDING SAMPLE H1 SITE H2 MUTATION SITEACTIVITY GpH7-A5/GpH7-HA7/GpL16-k0 A5 — HA7 S239D P329R 3 (SEQ ID NO: 3,34, 5) GpH7-A5/GpH7-HA15/GpL16-k0 A5 — HA15 A330L P329R 32 (SEQ ID NO:3, 35, 5) GpH7-A5/GpH7-HA18/GpL16-k0 A5 — HA18 I332E P329R 35 (SEQ IDNO: 3, 36, 5) GpH7-A5/GpH7-HA5/GpL16-k0 A5 — HA5 L234Y P329R 60 (SEQ IDNO: 3, 19, 5) GpH7-A5/GpH7-HA6/GpL16-k0 A5 — HA6 G236W P329R 56 (SEQ IDNO: 3, 20, 5) GpH7-A5/GpH7-HA11/GpL16-k0 A5 — HA11 S298A P329R 84 (SEQID NO: 3, 21, 5) GpH7-A48/GpH7-B3-06-09D/GpL16-k0 A48 P329R B3-06-09DS239D — 123 (SEQ ID NO: 16, 37, 5) GpH7-A48/GpH7-B3-20-08L/GpL16-k0 A48P329R B3-20-08L A330L — 60 (SEQ ID NO: 16, 38, 5)GpH7-A48/GpH7-B3-22-10E/GpL16-k0 A48 P329R B3-22-10E I332E — 189 (SEQ IDNO: 16, 39, 5) GpH7-A48/GpH7-B3-01-15Y/GpL16-k0 A48 P329R B3-01-15YL234Y — 11 (SEQ ID NO: 16, 22, 5) GpH7-A48/GpH7-B3-03-20W/GpL16-k0 A48P329R B3-03-20W G236W — 13 (SEQ ID NO: 16, 23, 5)GpH7-A48/6pH7-B3-15-02A/GpL16-k0 A48 P329R B3-15-02A S298A — 47 (SEQ IDNO: 16, 24, 5)

The column “SAMPLE” indicates antibody names; the columns “H1” and “H2”indicate names of the H chain constant region of respective antibodies;and the column “MUTATION SITE” indicates mutations that are different incomparison with GpH7-A5/GpH7-B3/GpL16-k0 (“-”: when there is noparticular mutation). The FcγRIIIa-binding activity is indicated as arelative binding activity when setting the FcγRIIIa binding ofGpH7-A5/GpH7-B3/GpL16-k0 as 100. The SEQ ID NOs are also shown for theamino acid sequences of the H chain and L chain of each antibody.

Based on the result, as to each altercation, the FcγRIIIa-bindingactivity is compared between when each alteration is introduced into thesame H chain as P329R, and when introduced into the different H chainfrom P329R. In the case of S239D, the binding activity ofGpH7-A5/GpH7-HA7/GpL16-k0 corresponding to the former was 3, while thebinding activity of GpH7-A48/GpH7-B3-06-09D/GpL16-k0 corresponding tothe latter was 123; thus, the FcγRIIIa binding was inhibited when thealteration was introduced into the same H chain as P329R. In the case ofA330L, the binding activity of GpH7-A5/GpH7-HA15/GpL16-k0 correspondingto the former was 32, while the binding activity ofGpH7-A48/GpH7-B3-20-08L/GpL16-k0 corresponding to the latter was 60;thus, the FcγRIIIa binding was inhibited when the alteration wasintroduced into the same H chain as P329R. In the case of I332E, thebinding activity of GpH7-A5/GpH7-HA18/GpL16-k0 corresponding to theformer was 35, while the binding activity ofGpH7-A48/GpH7-B3-22-10E/GpL16-k0 corresponding to the latter was 189;thus, the FcγRIIIa binding was inhibited when the alteration wasintroduced into the same H chain as P329R. As to all of the alterations,the FcγRIIIa binding was inhibited when the alteration was introducedinto the same H chain as P329R, i.e., the former case. If the H chainintroduced with P329R corresponded to the H_(A) chain of FIG. 23, P329Ris thought to inhibit the binding from the X-direction. Sincecombinations that significantly inhibit the binding are those whenS239D, A330L, or I332E was introduced into the same H chain as P329R;that is, in this case, the alterations were introduced into the H_(A)chain. Since the effect to enhance the FcγRIIIa binding was markedlyinhibited when any of the alterations S239D, A330L, and I332E wasintroduced into the same H chain as P329R, all the alterations, whenintroduced into the H_(A) chain, enhance the FcγRIIIa binding from theX-direction which is inhibited by P329R. Thus, it is thought that theFcγRIIIa binding can be further enhanced by introducing thesealterations into the same H chain. Based on Example 5, it was consideredthat all of L234Y, G236W, and S298A, when introduced into the H_(B)chain, enhance the binding from the X-direction. As discussed inExamples 5 and 6, one can find a method for appropriately combiningrespective alterations by combining them with P329R. Based on the aboveresults, to enhance the FcγRIIIa binding from the X-direction as shownin FIG. 3, S239D, A330L, and I332E need to be introduced into the H_(A)chain, whereas L234Y, G236W, and S298A need to be introduced into theH_(B) chain. Thus, it was thought that the FcγRIIIa binding from theX-direction can be further enhanced by introducing the respectivealteration groups into different H chains.

To assess the above hypothesis, expression vectors inserted with thefollowing were constructed according to the method described inReference Example 1:

GpH7-A57 (SEQ ID NO: 40) resulting from the introduction of all ofS239D, A330L, and I332E into GpH7-A5;

GpH7-B78 (SEQ ID NO: 41) resulting from the introduction of all ofS239D, A330L, and I332E into GpH7-B3;

GpH7-TA7 (SEQ ID NO: 31) resulting from the introduction of all ofL234Y, G236W, and S298A into GpH7-A5; and

GpH7-TA45 (SEQ ID NO: 32) resulting from the introduction of all ofL234Y, G236W, and

S298A into GpH7-B3. Using the expression vectors, and GpH7-A5, GpH7-B3,and GpL16-k0, the following antibodies were expressed and preparedaccording to the method described in Reference Example 1:

hetero GpH7-TA7/GpH7-B78/GpL16-k0 in which one of the H chains has beenintroduced with L234Y, G236W, and S298A, and the other has beenintroduced with S239D, A330L, and I332E;

GpH7-TA7/GpH7-B3/GpL16-k0 in which only one of the H chains has beenintroduced with L234Y, G236W, and S298A;

GpH7-TA7/GpH7-TA45/GpL16-k0 in which both of the H chains have beenintroduced with L234Y, G236W, and S298A;

GpH7-A5/GpH7-B78/GpL16-k0 in which only one of the H chains has beenintroduced with S239D, A330L, and I332E; and

GpH7-A57/GpH7-B78/GpL16-k0 in which both of the H chains have beenintroduced with S239D, A330L, and I332E. The prepared antibodies werecompared for the FcγRIIIa-binding activity using, as an indicator, KDfor FcγRIIIa that was determined according to the method described inReference Example 2. The assessment result on the effect of thecombination of L234Y, G236W, and S298A with S239D, A330L, and I332E issummarized in Table 13.

TABLE 13 KD KD SAMPLE H1 MUTATION SITE H2 MUTATION SITE KD (M) ratio 1ratio 2 GpH7-G1d/GpL16-k0 G1d G1d 1.2E−06 1 (SEQ ID NO: 2, 5)GpH7-A5/GpH7-B3/GpL16-k0 A5 — — — B3 — — — 1.6E−06 0.75 1 (SEQ ID NO: 3,4, 5) GpH7-TA7/GpH7-B3/GpL16-k0 TA7 L234Y G236W S298A B3 — — — 3.2E−073.8 5.1 (SEQ ID NO: 31, 4, 5) GpH7-A5/GpH7-B78/GpL16-k0 A5 — — — B78S239D A330L I332E 5.4E−08 23 30 (SEQ ID NO: 3, 41, 5)GpH7-A57/GpH7-B78/GpL16-k0 A57 S239D A330L I332E B78 S239D A330L I332E6.2E−09 199 263 (SEQ ID NO: 40, 41, 5) GpH7-TA7/GpH7-TA45/ TA7 L234YG236W S298A TA45 L234Y G236W S298A 3.3E−06 0.37 0.49 GpL16-k0 (SEQ IDNO: 31, 32, 5) GpH7-TA7/GpH7-B78/GpL16-k0 TA7 L234Y G236W S298A B78S239D A330L I332E 4.7E−09 261 347 (SEQ ID NO: 31, 41, 5)

The column “SAMPLE” indicates antibody names; the columns “H1” and “H2”indicate names of the H chain constant region of respective antibodies;and the column “MUTATION SITE” indicates mutations that are different incomparison with GpH7-A5/GpH7-B3/GpL16-k0 (“-”: when there is noparticular mutation). A value obtained by dividing KD ofGpH7-G1d/GpL16-k0 for FcγRIIIa by the KD of each antibody is defined as“KD ratio 1”, while a value obtained by dividing KD ofGpH7-A5/GpH7-B3/GpL16-k0 for FcγRIIIa by the KD of each antibody wasdefined as “KD ratio 2”. The SEQ ID NOs are also shown for the aminoacid sequences of the H chain and L chain of each antibody.

In view of the result of Table 13, when GpH7-A5/GpH7-B3/GpL16-k0 inwhich D356K, H435R, and K439E were each introduced into one H chain iscompared to GpH7-G1d/GpL16-k0 which is a native IgG1, the difference inthe FcγRIIIa-binding activity is 0.75 times, and no significantdifference was observed. Thus, it was thought that the alterationsD356K, H435R, and K439E do not affect the FcγRIIIa-binding activity.

Each alteration was assessed for its effect on homodimerized antibodiesthat use the prior art. The FcγRIIIa-binding activity of thehomodimerized antibody GpH7-A57/GpH7-B78/GpL16-k0 in which both of the Hchains were introduced with S239D, A330L, and I332E was increased byabout 260 times that of GpH7-A5/GpH7-B3/GpL16-k0. In contrast, theFcγRIIIa-binding activity of the homodimerized antibodyGpH7-TA7/GpH7-TA45/GpL16-k0 in which both of the H chains was introducedwith L234Y, G236W, and S298A was decreased to 0.49 times. Thisdemonstrates that only the group of the alterations S239D, A330L, andI332E has the effect to enhance the FcγRIIIa-binding activity ofhomodimerized antibodies.

Heterodimerized antibodies in which only one of the H chains wasintroduced with each alteration group were assessed for the effect ofeach alteration group. The FcγRIIIa-binding activity of theheterodimerized antibody GpH7-A5/GpH7-B78/GpL16-k0 in which one of the Hchains was introduced with S239D, A330L, and I332E was increased byabout 30 times that of GpH7-A5/GpH7-B3/GpL16-k0, while theFcγRIIIa-binding activity of the heterodimerized antibodyGpH7-TA7/GpH7-B3/GpL16-k0 in which one of the H chains was introducedwith L234Y, G236W, and S298A was increased by 5.1 times. This resultdemonstrates that the group of the alterations S239D, A330L, and I332Ehas the stronger effect to increase the FcγRIIIa-binding activity.

Each alteration group was assessed for the difference in its effectbetween the homodimerized antibody and heterodimerized antibody.Regarding S239D, A330L, and I332E, the FcγRIIIa-binding activity of theheterodimerized antibody was increased by 30 times that ofGpH7-A5/GpH7-B3/GpL16-k0, while the activity of the homodimerizedantibody was increased by about 260 times, demonstrating that thealterations, when introduced into homodimerized antibodies, furtherincrease the FcγRIIIa-binding activity. Meanwhile, regarding L234Y,G236W, and S298A, the FcγRIIIa-binding activity of the heterodimerizedantibody was increased by 5.1 times that of GpH7-A5/GpH7-B3/GpL16-k0;nevertheless, the activity of the homodimerized antibody was decreasedto 0.49 times. This result shows that the group of the alterationsL234Y, G236W, and S298A, only in heterodimerized antibodies, has theeffect to enhance the FcγRIIIa-binding activity, as discussed in Example5.

It was demonstrated that, in homodimerized antibodies, only the group ofthe alterations S239D, A330L, and I332E has the effect to enhance theFcγRIIIa binding, and also in heterodimerized antibodies, the group ofthe alterations S239D, A330L, and I332E more strongly enhances theFcγRIIIa binding. Based on the conventional concept, if the combinationof the group of the alterations S239D, A330L, and I332E with the groupof the alterations L234Y, G236W is considered, it would be predictedthat the effect to enhance the FcγRIIIa binding is the strongest in thehomodimerized antibody GpH7-A57/GpH7-B78/GpL16-k0 in which both of the Hchains are introduced with only the group of the alterations S239D,A330L, and I332E, which strongly enhance the FcγRIIIa binding of bothheterodimerized antibody and homodimerized antibody. However, theFcγRIIIa-binding activity of the heterodimerized antibodyGpH7-TA7/GpH7-B78/GpL16-k0 in which S239D, A330L, and I332E wereintroduced into one H chain and L234Y, G236W, and S298A were introducedinto the other H chain was increased by about 350 times that ofGpH7-A5/GpH7-B3/GpL16-k0; the effect to enhance the binding was strongerthan that for the homodimerized antibody in which both of the H chainswere introduced with S239D, A330L, and I332E. This supports thehypothesis that, when the group of the alterations S239D, A330L, andI332E, and the group of the alterations L234Y, G236W, and S298A areintroduced into different H chains, all of the introduced alterationsenhance the FcγRIIIa-binding activity in both the H_(A) chain and H_(B)chain, and the effect is greater than the case when the group of thealterations S239D, A330L, and I332E are introduced into both H chains.

That is, it is demonstrated that the use of heterodimerized antibodies,instead of conventional homodimerized antibodies, allows fineroptimization of the asymmetric interaction between the Fc domain andFcγRIIIa to design Fc domains having stronger binding activity.

In view of FIG. 23, it was thought that A330L and I332E interact withFcγR only in the H_(A) chain, while S239D interacts with FcγR in boththe H_(A) chain and H_(B) chain. Indeed, KD for FcγRIIIa of theheterodimerized antibody GpH7-A5/GpH7-B78/GpL16-k0 in which only one ofthe H chains was introduced with S239D, A330L, and I332E was 5.4E-8,while the KD of the homodimerized antibody GpH7-A57/GpH7-B78/GpL16-k0was 6.2E-9; thus, the FcγRIIIa-binding activity was increased by 8.7times. If it is thought that the difference in the binding activity isdue to the involvement of S239D in the enhancement of the binding inboth H chains, this difference is thought to be eliminated byintroducing S239D into both H chains. To assess this hypothesis,GpH7-A53 (SEQ ID NO: 42) resulting from the introduction of S239D intoGpH7-A5 was constructed, and, this is combined with GpH7-B78 introducedwith S239D, A330L, and I332E, and the expression and preparation wereperformed according to the method described in Reference Example 1. Theeffect of the combination of S239D and S239D, A330L, and I332E wasassessed by comparing the FcγRIIIa binding between the heterodimerizedand homodimerized antibodies with S239D, A330L, and I332E according tothe method described in Reference Example 2 (Table 14).

TABLE 14 KD Sample H1 MUTATION SITE H2 MUTATION SITE KD (M) ratioGpH7-A5/GpH7-B78/GpL16-k0 A5 — — — B78 S239D A330L I332E 5.4E−08 1 (SEQID NO: 3, 41, 5) GpH7-A53/GpH7-B78/GpL16-k0 A53 S239D — — B78 S239DA330L I332E 1.1E−08 4.9 (SEQ ID NO: 42, 41, 5)GpH7-A57/GpH7-B78/GpL16-k0 A57 S239D A330L I332E B78 S239D A330L I332E6.2E−09 8.7 (SEQ ID NO: 40, 41, 5)

The column “SAMPLE” indicates antibody names; the columns “H1” and “H2”indicate names of the H chain constant region of respective antibodies;and the column “MUTATION SITE” indicates mutations that are different incomparison with GpH7-A5/GpH7-B3/GpL16-k0 (“-”: when there is noparticular mutation). A value obtained by dividing KD ofGpH7-A5/GpH7-B78/GpL16-k0 for FcγRIIIa by the KD of each antibody wasdefined as “KD ratio”. The SEQ ID NOs are also shown for the amino acidsequences of the H chain and L chain of each antibody.

Table 14 shows that the FcγRIIIa-binding activity ofGpH7-A53/GpH7-B78/GpL16-k0 in which one of the H chains was introducedwith S239D, A330L, and I332E and the other was introduced with S239D wasincreased by 4.9 times that of GpH7-A5/GpH7-B78/GpL16-k0 in which onlyone of the H chains was introduced with S239D, A330L, and I332E; whilethe activity was decreased by only 1.8 times that ofGpH7-A57/GpH7-B78/GpL16-k0 in which both of the H chains were introducedwith S239D, A330L, and I332E. This result demonstrates that S239Dinteracts with FcγRIIIa in both H chains, as mentioned above for thehypothesis. It was thought that the interaction with FcγRIIIa can befurther enhanced by introducing the above alteration.

The present inventors tested whether the FcγRIIIa-binding activity canbe further increased by introducing S239D into the heterodimerizedantibody GpH7-TA7/GpH7-B78/GpL16-k0 in which one of the H chains wasintroduced with L234Y, G236W, and S298A and the other was introducedwith S239D, A330L, and I332E. GpH7-TA22 (SEQ ID NO: 43) was constructedby introducing S239D into GpH7-TA7, and then inserted into an expressionvector; and the resulting expression vector was combined with GpL16-k0,and GpH7-B78 obtained by introducing S239D, A330L, and I332E intoGpH7-B3; and the antibody of interest was expressed and preparedaccording to the method described in Reference Example 1. Furthermore,the heterodimerized antibody GpH7-TA22/GpH7-B78/GpL16-k0 was prepared byintroducing L234Y, G236W, S239D, and S298A into one H chain, and S239D,A330L, and I332E into the other H chain. To assess the effect of thecombination of S239D with S239D, A330L, and I332E, the antibodies werecompared for the FcγRIIIa-binding activity according to the methoddescribed in Reference Example 2 (Table 15).

TABLE 15 KD Sample H1 MUTATION SITE H2 MUTATION SITE KD (M) ratioGpH7-TA7/GpH7-B78/GpL16-k0 TA7 L234Y/G236W/S298A — B78 S239D/A330L/I332E4.1E−09 1 (SEQ ID NO: 31, 41, 5) GpH7-TA22/GpH7-B78/GpL16-k0 TA22L234Y/G236W/S298A S239D B78 S239D/A330L/I332E 1.3E−09 3.2 (SEQ ID NO:43, 41, 5)

The column “SAMPLE” indicates antibody names; the columns “H1” and “H2”indicate names of the H chain constant region of respective antibodies;and the column “MUTATION SITE” indicates mutations that are different incomparison with GpH7-A5/GpH7-B3/GpL16-k0 (“-”: when there is noparticular mutation). A value obtained by dividing KD ofGpH7-TA7/GpH7-B78/GpL16-k0 for FcγRIIIa by the KD of each antibody wasdefined as “KD ratio”. The SEQ ID NOs are also shown for the amino acidsequences of the H chain and L chain of each antibody.

Table 15 shows that the binding activity of GpH7-TA22/GpH7-B78/GpL16-k0resulting from the introduction of S239D into the H chain that does notcontain S239D in the heterodimerized antibody GpH7-TA7/GpH7-B78/GpL16-k0in which one of the H chains was introduced with L234Y, G236W, and S298Aand the other H chain with S239D, A330L, and I332E was increased by 3.2times that of GpH7-TA7/GpH7-B78/GpL16-k0. This result demonstrates thatthe FcγRIIIa binding can be further enhanced by using S239D.

Then, the present inventors considered further introducing thealterations Y296W and K334G that enhance the FcγRIIIa binding, whichwere revealed as described in Example 4.

First, it was considered which H chain should be introduced with Y296W.GpH7-TA52 (SEQ ID NO: 44) resulting from the introduction of themutation Y296W into GpH7-TA7 was constructed, and expressed and preparedin combination with GpH7-B78 according to the method described inReference Example 1. Furthermore, GpH7-TA58 (SEQ ID NO: 45) resultingfrom the introduction of Y296W into GpH7-B78 was constructed, andexpressed and prepared in combination with GpH7-TA22 according to themethod described in Reference Example 1. To assess the effects ofcombinations with Y296W, the prepared antibodies were compared for theFcγRIIIa-binding activity according to the method described in ReferenceExample 2 (Table 16).

TABLE 16 KD Sample H1 MUTATION SITE H2 MUTATION SITE KD (M) ratioGpH7-TA7/GpH7-B78/GpL16-k0 TA7 L234Y/G236W/S298A — B78 S239D/A330L/I332E— 4.1E−09 1 (SEQ ID NO: 31, 41, 5) GpH7-TA7/GpH7-TA58/GpL16-k0 TA7L234Y/G236W/S298A — TA58 S239D/A330L/I332E Y296W 3.3E−09 1.2 (SEQ ID NO:31, 45, 5) GpH7-TA52/GpH7-B78/GpL16-k0 TA52 L234Y/G236W/S298A Y296W B78S239D/A330L/I332E — 2.3E−09 1.8 (SEQ ID NO: 44, 41, 5)

The column “Sample” indicates antibody names; the columns “H1” and “H2”indicate names of H chain constant region of respective antibodies; andthe column “MUTATION SITE” indicates mutations that are different incomparison with GpH7-A5/GpH7-B3/GpL16-k0 (“-”: when there is noparticular mutation). A value obtained by dividing KD ofGpH7-TA7/GpH7-B78/GpL16-k0 for FcγRIIIa by the KD of each antibody wasdefined as “KD ratio”. The SEQ ID NOs are also shown for the amino acidsequences of the H chain and L chain of each antibody.

The result showed that Y296W, when introduced into the H chain differentfrom that introduced with L234Y, G236W, and S298A, increased theFcγRIIIa-binding activity only by 1.2 times after introduction comparedto before; while Y296W, when introduced into the same H chain, increasedthe FcγRIIIa-binding activity by 1.8 times that ofGpH7-TA7/GpH7-B78/GpL16-k0. From this result, it is thought that Y296W,when introduced into the same H chain as L234Y, G236W, and S298A, hasthe effect to enhance the FcγRIIIa binding.

Then, GpH7-TA54 (SEQ ID NO: 46) resulting from the introduction of Y296Winto GpH7-TA22 was constructed, and expressed and prepared incombination with GpH7-B78 according to the method described in ReferenceExample 1. Furthermore, GpH7-TA58 (SEQ ID NO: 45) resulting from theintroduction of Y296W into GpH7-B78 was constructed, and expressed andprepared in combination with GpH7-TA22 according to the method describedin Reference Example 1. To assess the effect of combination with Y296W,the prepared antibodies were compared for the FcγRIIIa-binding activityaccording to the method described in Reference Example 2 (Table 17).

TABLE 17 Sample H1 MUTATION SITE H2 MUTATION SITE KD (M) KD ratioGpH7-TA22/GpH7-B78/GpL16-k0 TA22 L234Y/G236W/S239D/S298A — B78S239D/A330L/I332E — 1.3E−09 1 (SEQ ID NO: 43, 41, 5)GpH7-TA22/GpH7-TA58/GpL16-k0 TA22 L234Y/G236W/S239D/S298A — TA58S239D/A330L/I332E Y296W 1.3E−09 1.0 (SEQ ID NO: 43, 45, 5)GpH7-TA54/GpH7-B78/GpL16-k0 TA54 L234Y/G236W/S239D/S298A Y296W B78S239D/A330L/I332E — 1.0E−09 1.3 (SEQ ID NO: 46, 41, 5)

The column “Sample” indicates antibody names; the columns “H1” and “H2”indicate names of the H chain constant region of respective antibodies;and the column “MUTATION SITE” indicates mutations that are different incomparison with GpH7-A5/GpH7-B3/GpL16-k0 (“-”: when there is noparticular mutation). A value obtained by dividing KD ofGpH7-TA22/GpH7-B78/GpL16-k0 for FcγRIIIa by the KD of each antibody wasdefined as “KD ratio”. The SEQ ID NOs are also shown for the amino acidsequences of the H chain and L chain of each antibody.

The result shown in Table 17 demonstrates that, when Y296W wasintroduced into the H chain that is different from that introduced withL234Y, G236W, and S298A, there was no difference in the FcγRIIIa-bindingactivity after the introduction compared to before. When Y296W wasintroduced into the same H chain as L234Y, G236W, and S298A, theFcγRIIIa-binding activity was increased by 1.3 times that ofGpH7-TA22/GpH7-B78/GpL16-k0. From this result, it was thought thatY296W, when introduced into the same H chain as L234Y, G236W, and S298A,has the effect to enhance the FcγRIIIa-binding activity.

Then, K334G was also assessed in the same manner. GpH7-TA40 (SEQ ID NO:47) resulting from the introduction of K334G into GpH7-TA7 wasconstructed, and expressed and prepared in combination with GpH7-B78according to the method described in Reference Example 1. Furthermore,GpH7-TA50 (SEQ ID NO: 48) resulting from the introduction of K334G intoGpH7-B78 was constructed, and expressed and prepared in combination withGpH7-TA7 according to the method described in Reference Example 1. Toassess the effect of combinations with K334G, the prepared antibodieswere compared for the FcγRIIIa binding according to the method describedin Reference Example 2 (Table 18).

TABLE 18 KD Sample H1 MUTATION SITE H2 MUTATION SITE KD (M) ratioGpH7-TA7/GpH7-B78/GpL16-k0 TA7 L234Y/G236W/S298A — B78 S239D/A330L/I332E— 4.1E−09 1.0 (SEQ ID NO: 31, 41, 5) GpH7-TA7/GpH7-TA50/GpL16-k0 TA7L234Y/G236W/S298A — TA50 S239D/A330L/I332E K334G 3.4E−09 1.2 (SEQ ID NO:31, 48, 5) GpH7-TA40/GpH7-B78/GpL16-k0 TA40 L234Y/G236W/S298A K334G B78S239D/A330L/I332E — 7.5E−09 0.5 (SEQ ID NO: 47, 41, 5)

The column “Sample” indicates antibody names; the columns “H1” and “H2”indicate names of the H chain constant regions of each antibody; and thecolumn “MUTATION SITE” indicates mutations that are different incomparison with GpH7-A5/GpH7-B3/GpL16-k0 (“-”: when there is noparticular mutation). A value obtained by dividing KD ofGpH7-TA7/GpH7-B78/GpL16-k0 for FcγRIIIa by the KD of each antibody wasdefined as “KD ratio”. The SEQ ID NOs are also shown for the amino acidsequences of the H chain and L chain of each antibody.

The result shown in Table 18 demonstrates that, when K334G wasintroduced into the same H chain as L234Y, G236W, and S298A, theFcγRIIIa-binding activity is decreased by half after the introductioncompared to before. When K334G was introduced into the different Hchain, the FcγRIIIa-binding activity was increased by 1.2 times that ofGpH7-TA7/GpH7-B78/GpL16-k0. From this result, it was thought that K334G,when introduced into the H chain different from that introduced withL234Y, G236W, and S298A, has the effect to enhance the FcγRIIIa-bindingactivity.

[Example 8] Improvement of the Selectivity for Activating FcγR orInhibitory FcγR

There are activating FcγR which has ITAM, and inhibitory FcγR which hasITIM. Representative activating FcγRs (activating receptor) includeFcγRIa, FcγRIIa, and FcγRIIIa, while representative inhibitory FcγRs(inhibitory receptor) include FcγRIIb. Regarding antibodies targeted tocancer, the ratio of the binding activity to activating FcγR whoseaction mechanism is based on ADCC activity or antibody-dependentcellular phagocytosis (ADCP) activity against the binding activity toinhibitory FcγR is believed to play an important role (Nature Medicine,6: 443-446, 2000).

For antibodies targeted to cancer, it is desirable to increase theirbinding activity to activating FcγR while reducing their bindingactivity to inhibitory FcγR. Specifically, desirable alterations are,such as those comprised in Region a shown in FIG. 24, alterations thatallow an antibody to bind to activating FcγR more strongly than thenative antibody, and bind to inhibitory FcγR more weakly than the nativeantibody, i.e., alterations that enhance the binding in an activatingFcγR-selective manner. Also, the desirable alterations are, such asthose in Region b shown in FIG. 25, alterations that allow the ratiobetween the binding activity to activating FcγR and the binding activityto inhibitory FcγR to be greater than the native antibody. It can besaid that such alterations selectively increase the binding activity toactivating FcγR as compared to inhibitory FcγR.

The heterodimerized antibodies He Abs in which one of the H chains wasintroduced with an alteration were assayed for their binding activitiesto each activating FcγR and inhibitory FcγR according to the methoddescribed in Example 4. The result is summarized in FIGS. 26, 27, 28 and29 to assess the ratio of the binding activity to each activating FcγRand inhibitory FcγR for each heterodimerized antibody. The activatingFcγRs in FIGS. 26, 27, 28, and 29 are FcγRIa, FcγRIIa (R), FcγRIIa (H),and FcγRIIIa, respectively.

The alterations present in the region in FIG. 26 corresponding to a andb in FIGS. 24 and 25 are listed in Table 19 (Table 19-1 to 19-5).Likewise, as to FcγRIIa (R) (FIG. 27), FcγRIIa (H) (FIG. 28), FcγRIIIa(FIG. 29), alterations present in the region corresponding to a and bare listed in Table 20 (Table 20-1 to 20-3), Table 21 (Table 21-1 to21-3), and Table 22 (Table 22-1 to 22-3).

TABLE 19-1 REGION a Ia > Con REGION b IIb < Con Ia/Iib=>1.2 Ia bindingIIb binding Ia binding IIb binding Ia/IIb NAME He/Con_1a He/Con_2b NAMEHe/Con_1a He/Con_2b He_1a/2b L234_03V 104.4 73.1 L234_01G 96.1 50.7 1.90L234_04F 101.0 99.8 L234_02A 97.5 64.9 1.50 L234_06M 103.8 78.5 L234_03V104.4 73.1 1.43 L234_07I 102.9 80.9 L234_05P 99.9 72.9 1.37 L234_15Y100.1 96.3 L234_06M 103.8 78.5 1.32 G236_02A 100.2 96.5 L234_07I 102.980.9 1.27 G236_20W 105.2 58.2 L234_11K 91.8 16.2 5.67 S239_14T 101.197.3 L234_12R 91.8 18.9 4.86 S239_19Q 100.6 86.1 L234_13S 96.9 59.3 1.63D265_10E 101.4 38.2 L234_14T 98.6 62.5 1.58 V266_19Q 100.2 89.2 L234_16H94.0 59.4 1.58 S267_16H 100.0 65.9 L234_19Q 95.7 60.3 1.59 S267_18N102.4 47.7 L235_01G 87.2 35.4 2.46 H268_04F 106.3 89.6 L235_02A 92.263.0 1.46 H268_06M 104.7 65.7 L235_03V 94.7 66.4 1.43 H268_07I 107.888.9 L235_05P 94.7 54.7 1.73 H268_08L 107.4 67.8 L235_06M 98.0 81.0 1.21H268_11K 105.8 83.0 L235_11K 79.9 6.3 12.73 H268_12R 106.1 89.8 L235_12R78.0 9.6 8.12 H268_14T 112.2 86.0 L235_13S 90.5 48.2 1.88 E269_01G 104.451.5 L235_14T 88.7 46.8 1.90 E269_02A 104.4 49.9 L235_18N 92.2 54.9 1.68E269_03V 102.6 45.1 L235_19Q 94.0 42.6 2.21 E269_04F 103.1 45.0 G236_03V89.9 35.3 2.55 E269_05P 102.3 52.6 G236_04F 96.4 39.3 2.45 E269_06M103.6 46.7 G236_05P 77.0 19.0 4.05 E269_07I 102.8 43.9 G236_06M 96.745.5 2.13 E269_08L 103.6 42.9 G236_07I 87.5 31.0 2.82 E269_13S 103.147.8 G236_08L 79.6 26.8 2.97 E269_14T 105.9 50.6 G236_11K 71.0 49.8 1.42E269_15Y 106.4 44.7 G236_12R 63.1 6.8 9.30 E269_16H 105.8 38.5 G236_14T92.2 57.8 1.59 E269_18N 101.5 44.8 G236_15Y 92.5 39.5 2.34 E269_19Q105.0 44.3 G236_16H 83.5 27.9 2.99 E269_20W 105.2 42.5 G236_19Q 92.146.6 1.98 P271_02A 103.5 88.0 G236_20W 105.2 58.2 1.81 P271_03V 102.374.8 G237_03V 78.5 61.4 1.28 P271_04F 101.8 59.5 G237_05P 44.5 24.6 1.81P271_06M 102.5 77.5 G237_11K 63.3 9.0 7.05 P271_07I 102.5 76.8 G237_12R65.8 9.8 6.71 P271_11K 102.8 90.2 G237_14T 82.3 54.8 1.50 P271_12R 102.486.5 G237_16H 72.6 55.2 1.32 P271_13S 102.4 87.6 P238_01G 92.9 56.0 1.66P271_14T 102.3 95.4 P238_02A 95.2 57.4 1.66 P271_15Y 103.0 52.3 P238_07I99.1 80.0 1.24 P271_16H 102.1 67.9 P238_11K 77.4 18.1 4.27 P271_18N103.2 93.3 P238_12R 77.3 51.4 1.50 P271_19Q 103.1 88.6 P238_13S 96.670.4 1.37 P271_20W 102.6 58.1 P238_14T 91.1 50.7 1.80 Q295_02A 104.279.7 P238_18N 93.3 73.2 1.27 Q295_03V 104.1 75.7 S239_04F 93.8 44.1 2.13Q295_04F 102.2 64.7 S239_11K 68.0 24.7 2.75 Q295_05P 103.4 67.5 S239_12R86.5 39.1 2.21 Q295_06M 104.2 90.4 S239_15Y 89.8 34.9 2.57 Q295_07I103.0 84.3 S239_16H 88.1 49.4 1.78

TABLE 19-2 REGION a Ia > Con REGION b IIb < Con Ia/Iib=>1.2 Ia bindingIIb binding Ia binding IIb binding Ia/IIb NAME He/Con_1a He/Con_2b NAMEHe/Con_1a He/Con_2b He_1a/2b Q295_09D 100.1 60.8 S239_20W 92.7 45.1 2.06Q295_10E 103.1 94.8 D265_01G 79.9 16.1 4.95 Q295_11K 101.3 70.4 D265_02A84.5 19.9 4.25 Q295_12R 100.4 65.2 D265_03V 55.7 3.2 17.30 Q295_13S102.0 56.9 D265_04F 53.0 3.2 16.77 Q295_14T 102.7 66.8 D265_06M 75.710.0 7.57 Q295_15Y 101.7 65.4 D265_07I 54.4 1.3 41.18 Q295_16H 102.562.7 D265_08L 56.8 4.6 12.43 Q295_18N 102.4 64.9 D265_10E 101.4 38.22.66 Y296_01G 102.6 79.3 D265_11K 43.2 14.9 2.90 Y296_02A 102.9 82.0D265_12R 44.3 15.0 2.96 Y296_03V 104.0 79.3 D265_13S 68.7 11.3 6.07Y296_04F 104.1 99.2 D265_14T 78.5 16.0 4.90 Y296_06M 104.0 87.8 D265_15Y44.3 0.6 73.46 Y296_07I 104.3 84.3 D265_16H 85.4 38.3 2.23 Y296_08L103.9 82.3 D265_18N 27.7 6.1 4.52 Y296_09D 105.9 98.9 D265_19Q 88.8 33.42.66 Y296_10E 105.8 98.0 D265_20W 51.5 1.1 45.61 Y296_11K 104.7 72.8V266_01G 91.3 51.5 1.77 Y296_12R 106.3 79.5 V266_04F 96.7 57.6 1.68Y296_13S 105.1 86.3 V266_05P 71.9 0.4 205.21 Y296_14T 105.1 88.2V266_09D 79.3 10.1 7.88 Y296_16H 105.8 92.2 V266_10E 81.9 15.2 5.37Y296_18N 105.6 91.5 V266_11K 64.4 1.0 63.53 Y296_19Q 111.6 89.3 V266_13S90.6 54.4 1.67 S298_01G 103.6 80.1 V266_15Y 93.3 49.4 1.89 S298_02A106.6 73.4 V266_16H 89.1 57.2 1.56 S298_03V 109.7 64.5 V266_18N 99.569.7 1.43 S298_04F 109.0 65.4 V266_20W 91.4 71.9 1.27 S298_07I 107.070.6 S267_04F 97.7 48.0 2.04 S298_09D 105.9 48.3 S267_05P 91.8 31.5 2.91S298_10E 108.2 47.6 S267_11K 93.3 12.2 7.65 S298_11K 109.3 61.3 S267_12R92.7 11.3 8.21 S298_12R 109.4 57.8 S267_15Y 94.2 37.9 2.48 S298_14T107.1 77.9 S267_16H 100.0 65.9 1.52 S298_15Y 107.2 61.4 S267_18N 102.447.7 2.15 S298_16H 108.8 63.5 S267_20W 99.4 57.2 1.74 S298_18N 108.148.1 H268_06M 104.7 65.7 1.59 S298_19Q 109.4 73.3 H268_07I 107.8 88.91.21 S298_20W 104.0 41.1 H268_08L 107.4 67.8 1.58 Y300_01G 116.1 48.4H268_11K 105.8 83.0 1.28 Y300_02A 112.2 69.2 H268_14T 112.2 86.0 1.30Y300_03V 117.0 73.9 E269_01G 104.4 51.5 2.03 Y300_06M 112.1 84.3E269_02A 104.4 49.9 2.09 Y300_07I 107.8 74.5 E269_03V 102.6 45.1 2.27Y300_08L 113.0 73.6 E269_04F 103.1 45.0 2.29 Y300_09D 112.5 93.2E269_05P 102.3 52.6 1.94 Y300_11K 106.9 51.6 E269_06M 103.6 46.7 2.22Y300_12R 104.6 41.2 E269_07I 102.8 43.9 2.34 Y300_13S 111.1 53.9E269_08L 103.6 42.9 2.42 Y300_14T 111.7 55.6 E269_11K 95.2 17.8 5.34Y300_16H 110.9 87.0 E269_12R 97.1 25.2 3.86 Y300_20W 112.4 84.1 E269_13S103.1 47.8 2.16 K326_12R 100.1 89.2 E269_14T 105.9 50.6 2.09 S324_02A102.7 78.3 E269_15Y 106.4 44.7 2.38 S324_03V 103.6 97.8 E269_16H 105.838.5 2.75 S324_07I 102.0 95.7 E269_18N 101.5 44.8 2.27

TABLE 19-3 REGION a Ia > Con REGION b IIb < Con Ia/Iib=>1.2 Ia bindingIIb binding Ia binding IIb binding Ia/IIb NAME He/Con_1a He/Con_2b NAMEHe/Con_1a He/Con_2b He_1a/2b S324_08L 103.2 89.6 E269_19Q 105.0 44.32.37 S324_09D 103.5 85.8 E269_20W 105.2 42.5 2.47 S324_10E 104.9 77.4P271_03V 102.3 74.8 1.37 S324_11K 102.1 68.6 P271_04F 101.8 59.5 1.71S324_12R 101.7 63.1 P271_06M 102.5 77.5 1.32 S324_14T 120.9 88.4P271_07I 102.5 76.8 1.33 S324_15Y 113.8 88.2 P271_15Y 103.0 52.3 1.97S324_16H 112.6 83.5 P271_16H 102.1 67.9 1.50 S324_18N 115.5 81.1P271_20W 102.6 58.1 1.77 S324_19Q 112.3 66.8 D270_01G 76.3 30.5 2.50S324_20W 114.3 87.5 D270_02A 71.5 48.3 1.48 A327_03V 103.2 94.7 D270_03V77.9 39.5 1.97 A327_06M 103.2 59.5 D270_04F 78.1 53.1 1.47 A327_13S102.4 79.5 D270_05P 67.1 13.9 4.81 P329_01G 100.9 41.7 D270_06M 78.848.2 1.64 P329_02A 102.3 40.5 D270_07I 74.6 41.5 1.80 A330_03V 105.552.4 D270_08L 73.5 53.6 1.37 A330_04F 106.1 92.8 D270_11K 58.4 20.3 2.88A330_06M 104.6 80.5 D270_12R 66.3 18.3 3.63 A330_07I 105.2 67.1 D270_13S75.3 44.2 1.70 A330_08L 106.9 70.5 D270_14T 70.6 57.8 1.22 A330_09D108.6 55.6 D270_15Y 68.5 44.9 1.53 A330_10E 107.4 70.1 D270_16H 66.047.2 1.40 A330_11K 103.4 95.0 D270_18N 67.8 40.3 1.68 A330_14T 105.177.9 D270_19Q 78.2 48.0 1.63 A330_15Y 105.0 95.0 D270_20W 68.0 34.7 1.96A330_16H 105.8 87.6 Q295_01G 97.6 46.0 2.12 A330_18N 106.5 63.7 Q295_02A104.2 79.7 1.31 A330_19Q 105.2 90.8 Q295_03V 104.1 75.7 1.37 A330_20W107.6 76.1 Q295_04F 102.2 64.7 1.58 I332_12R 100.1 48.9 Q295_05P 103.467.5 1.53 E333_01G 102.6 81.4 Q295_07I 103.0 84.3 1.22 E333_02A 103.283.8 Q295_09D 100.1 60.8 1.65 E333_03V 105.0 93.2 Q295_11K 101.3 70.41.44 E333_04F 102.4 94.6 Q295_12R 100.4 65.2 1.54 E333_05P 102.3 97.7Q295_13S 102.0 56.9 1.79 E333_06M 102.1 85.4 Q295_14T 102.7 66.8 1.54E333_07I 102.0 96.2 Q295_15Y 101.7 65.4 1.56 E333_08L 102.2 95.0Q295_16H 102.5 62.7 1.64 E333_09D 103.5 94.3 Q295_18N 102.4 64.9 1.58E333_11K 102.1 80.6 Q295_20W 93.7 44.4 2.11 E333_12R 102.2 80.4 Y296_01G102.6 79.3 1.29 E333_13S 103.1 81.8 Y296_02A 102.9 82.0 1.26 E333_14T102.1 86.1 Y296_03V 104.0 79.3 1.31 E333_15Y 101.2 94.4 Y296_05P 95.636.6 2.61 E333_16H 100.8 83.3 Y296_07I 104.3 84.3 1.24 K334_01G 103.592.5 Y296_08L 103.9 82.3 1.26 K334_09D 106.5 94.8 Y296_11K 104.7 72.81.44 T335_01G 104.4 96.7 Y296_12R 106.3 79.5 1.34 T335_02A 103.3 94.0Y296_13S 105.1 86.3 1.22 T335_05P 106.4 99.9 Y296_19Q 111.6 89.3 1.25T335_06M 106.7 99.4 S298_01G 103.6 80.1 1.29 T335_11K 110.8 91.0S298_02A 106.6 73.4 1.45 T335_12R 112.8 91.9 S298_03V 109.7 64.5 1.70I336_01G 108.7 90.1 S298_04F 109.0 65.4 1.67 I336_02A 106.0 84.9S298_05P 91.6 11.9 7.70 I336_03V 105.2 90.3 S298_07I 107.0 70.6 1.52

TABLE 19-4 REGION a Ia > Con REGION b IIb < Con Ia/Iib=>1.2 Ia bindingIIb binding Ia binding IIb binding Ia/IIb NAME He/Con_1a He/Con_2b NAMEHe/Con_1a He/Con_2b He_1a/2b I336_04F 105.5 91.2 S298_09D 105.9 43.32.19 I336_09D 103.6 92.1 S298_10E 108.2 47.6 2.28 I336_10E 107.8 89.8S298_11K 109.3 61.3 1.78 I336_11K 105.4 90.3 S298_12R 109.4 57.8 1.89I336_12R 102.0 92.1 S298_14T 107.1 77.9 1.37 I336_13S 102.5 83.7S298_15Y 107.2 61.4 1.75 I336_14T 104.5 89.5 S298_16H 108.8 63.5 1.71I336_15Y 102.9 68.2 S298_18N 108.1 48.1 2.25 I336_16H 101.4 85.8S298_19Q 109.4 73.3 1.49 I336_18N 107.8 97.6 S298_20W 104.0 41.1 2.53I336_19Q 103.5 58.2 Y300_01G 116.1 48.4 2.40 S337_01G 106.6 92.0Y300_02A 112.2 69.2 1.62 S337_02A 105.8 97.2 Y300_03V 117.0 73.9 1.58S337_03V 106.7 98.9 Y300_05P 60.2 7.3 8.22 S337_04F 106.1 97.8 Y300_06M112.1 84.3 1.33 S337_06M 106.9 96.7 Y300_07I 107.8 74.5 1.45 S337_11K104.0 94.3 Y300_08L 113.0 73.6 1.54 S337_12R 105.8 99.5 Y300_09D 112.593.2 1.21 S337_19Q 104.6 99.7 Y300_11K 106.9 51.6 2.07 Y300_12R 104.641.2 2.54 Y300_13S 111.1 53.9 2.06 Y300_14T 111.7 55.6 2.01 Y300_16H110.9 87.0 1.27 Y300_20W 112.4 84.1 1.34 N325_02A 96.8 64.4 1.50N325_05P 88.8 26.0 3.42 N325_11K 80.9 10.4 7.81 N325_12R 84.2 19.1 4.41N325_16H 95.1 70.1 1.36 N325_19Q 99.1 69.1 1.43 S324_01G 98.8 81.1 1.22S324_02A 102.7 78.3 1.31 S324_09D 103.5 85.8 1.21 S324_10E 104.9 77.41.35 S324_11K 102.1 68.6 1.49 S324_12R 101.7 63.1 1.61 S324_14T 120.988.4 1.37 S324_15Y 113.8 88.2 1.29 S324_16H 112.6 83.5 1.35 S324_18N115.5 81.1 1.42 S324_19Q 112.3 66.8 1.68 S324_20W 114.3 87.5 1.31A327_04F 99.1 63.7 1.56 A327_05P 99.3 69.3 1.43 A327_06M 103.2 59.5 1.74A327_08L 98.6 63.1 1.56 A327_11K 86.1 12.5 6.87 A327_12R 86.7 9.0 9.64A327_13S 102.4 79.5 1.29 A327_15Y 96.3 60.8 1.58 A327_16H 96.5 63.5 1.52A327_19Q 96.2 54.2 1.78 A327_20W 97.7 73.8 1.24 L328_01G 97.2 80.2 1.21L328_05P 92.2 65.1 1.42 L328_11K 79.2 7.5 10.52 L328_12R 78.6 9.2 8.51

TABLE 19-5 REGION b Ia/Iib=>1.2 Ia binding IIb binding Ia/IIb NAMEHe/Con_1a He/Con_2b He_1a/2b L328_18N 98.7 80.8 1.22 P329_01G 100.9 41.72.42 P329_02A 102.3 40.5 2.52 P329_03V 97.4 38.5 2.53 P329_04F 92.7 32.22.88 P329_06M 85.3 29.1 2.94 P329_07I 94.1 34.6 2.72 P329_08L 85.7 29.12.95 P329_09D 77.6 53.5 1.45 P329_10E 78.8 52.7 1.50 P329_11K 70.4 16.94.17 P329_12R 74.3 17.6 4.22 P329_13S 93.3 41.0 2.27 P329_14T 89.7 37.92.37 P329_15Y 95.6 45.7 2.09 P329_16H 95.6 39.1 2.45 P329_18N 92.4 40.42.29 P329_19Q 70.7 30.7 2.31 P329_20W 96.9 45.1 2.15 A330_03V 105.5 52.42.01 A330_06M 104.6 80.5 1.30 A330_07I 105.2 67.1 1.57 A330_08L 106.970.5 1.52 A330_09D 108.6 55.6 1.95 A330_10E 107.4 70.1 1.53 A330_13S91.5 41.3 2.22 A330_14T 105.1 77.9 1.35 A330_16H 105.8 87.6 1.21A330_18N 106.5 63.7 1.67 A330_20W 107.6 76.1 1.41 P331_01G 85.3 45.41.88 I332_12R 100.1 48.9 2.05 E333_01G 102.6 81.4 1.26 E333_02A 103.283.8 1.23 E333_11K 102.1 80.6 1.27 E333_12R 102.2 80.4 1.27 E333_13S103.1 81.8 1.26 E333_16H 100.8 83.3 1.21 E333_18N 96.6 76.1 1.27T335_11K 110.8 91.0 1.22 T335_12R 112.8 91.9 1.23 I336_01G 108.7 90.11.21 I336_02A 106.0 84.9 1.25 I336_13S 102.5 83.7 1.22 I336_15Y 102.968.2 1.51 I336_19Q 103.5 58.2 1.78 I336_20W 86.7 20.3 4.28

The table shows a list of alterations that selectively enhance thebinding to FcγRIa as compared to FcγRIIb.

TABLE 20-1 REGION a IIa > Con REGION b IIb < Con IIa/IIb=>1.2 IIaRbinding IIb binding IIaR binding IIb binding IIaR/IIb NAME He/Con_2aRHe/Con_2b NAME He/Con_2aR He/Con_2b He_2aR/2b L234_04F 104.0 99.8L234_01G 62.3 50.7 1.23 G236_02A 145.9 96.5 L234_02A 79.6 64.9 1.23G236_13S 136.4 96.7 L234_05P 89.6 72.9 1.23 G236_14T 102.7 57.8 L234_11K26.8 16.2 1.66 S239_14T 103.4 97.3 L234_12R 29.9 18.9 1.58 H268_11K100.4 83.0 L234_14T 77.5 62.5 1.24 H268_12R 104.7 89.8 L234_16H 71.859.4 1.21 Q295_06M 101.7 90.4 L234_19Q 75.1 60.3 1.24 Q295_10E 101.694.8 L235_01G 48.5 35.4 1.37 Y296_04F 109.4 99.2 L235_11K 10.8 6.3 1.72Y296_06M 101.2 87.8 L235_12R 16.2 9.6 1.69 Y296_07I 101.0 84.3 L235_13S65.3 48.2 1.36 Y296_09D 105.7 98.9 L235_14T 60.7 46.8 1.30 Y296_10E102.6 98.0 L235_16H 94.8 76.9 1.23 Y296_13S 101.9 86.3 L235_18N 66.554.9 1.21 Y296_14T 103.1 88.2 L235_19Q 56.2 42.6 1.32 Y296_16H 104.792.2 G236_02A 145.9 96.5 1.51 Y296_18N 104.1 91.5 G236_03V 91.6 35.32.60 Y296_19Q 102.3 89.3 G236_04F 53.1 39.3 1.35 Y300_04F 107.8 94.8G236_05P 47.9 19.0 2.52 Y300_18N 100.4 82.7 G236_06M 57.3 45.5 1.26K326_12R 102.9 89.2 G236_07I 87.1 31.0 2.81 N325_10E 104.7 95.9 G236_08L65.8 26.8 2.45 N325_14T 105.4 91.6 G236_12R 24.1 6.8 3.55 N325_15Y 100.799.5 G236_13S 136.4 96.7 1.41 S324_04F 104.0 89.1 G236_14T 102.7 57.81.78 A330_11K 110.1 95.0 G236_15Y 58.6 39.5 1.48 A330_12R 103.5 84.8G236_16H 62.7 27.9 2.24 A330_15Y 101.9 95.0 G236_19Q 74.2 46.6 1.59A330_19Q 104.1 90.8 G236_20W 77.9 58.2 1.34 P331_02A 105.9 99.5 G237_03V93.0 61.4 1.52 P331_08L 101.7 95.7 G237_05P 34.5 24.6 1.40 P331_09D102.2 94.3 G237_11K 17.1 9.0 1.90 P331_13S 104.7 96.8 G237_12R 24.2 9.82.47 P331_14T 104.3 99.8 G237_14T 78.6 54.8 1.43 P331_18N 104.6 99.2G237_16H 74.5 55.2 1.35 E333_04F 102.1 94.6 P238_01G 75.0 56.0 1.34E333_05P 104.8 97.7 P238_02A 75.1 57.4 1.31 E333_07I 104.4 96.2 P238_11K36.9 18.1 2.03 E333_08L 103.0 95.0 P238_12R 80.5 51.4 1.57 E333_09D103.1 94.3 P238_13S 88.9 70.4 1.26 E333_15Y 100.7 94.4 P238_14T 73.150.7 1.44 T335_01G 103.8 96.7 P238_18N 91.9 73.2 1.26 T335_02A 102.494.0 S239_04F 54.8 44.1 1.24 T335_05P 103.6 99.9 S239_11K 48.5 24.7 1.96T335_06M 104.7 99.4 S239_12R 53.9 39.1 1.38 T335_12R 100.2 91.9 S239_15Y42.2 34.9 1.21 I336_18N 104.1 97.6 S239_16H 69.2 49.4 1.40 S337_02A105.0 97.2 D265_01G 24.1 16.1 1.49 S337_03V 106.1 98.9 D265_02A 33.919.9 1.71 S337_04F 104.6 97.8 D265_03V 5.8 3.2 1.80 S337_06M 105.2 96.7D265_04F 5.6 3.2 1.79 S337_11K 102.9 94.3 D265_06M 15.8 10.0 1.58S337_12R 107.2 99.5 D265_07I 3.4 1.3 2.57 S337_19Q 105.6 99.7 D265_08L6.6 4.6 1.46

TABLE 20-2 REGION b IIa/IIb->1.2 IIaR binding IIb binding IIaR/IIb NAMEHe/Con_2aR He/Con_2b He_2aR/2b D265_10E 58.2 38.2 1.52 D265_11K 33.714.9 2.26 D265_12R 32.2 15.0 2.15 D265_13S 20.5 11.3 1.81 D265_14T 25.816.0 1.61 D265_15Y 4.6 0.6 7.57 D265_16H 58.0 38.3 1.51 D265_18N 12.46.1 2.02 D265_19Q 54.8 33.4 1.64 D265_20W 4.2 1.1 3.73 V266_12R −5.1−1.1 4.78 V266_16H 76.5 57.2 1.34 H268_06M 81.0 65.7 1.23 H268_08L 85.067.8 1.25 H268_11K 100.4 83.0 1.21 E269_01G 65.3 51.5 1.27 E269_02A 65.349.9 1.31 E269_03V 58.8 45.1 1.30 E269_04F 58.2 45.0 1.29 E269_06M 61.746.7 1.32 E269_07I 58.8 43.9 1.34 E269_08L 57.2 42.9 1.33 E269_11K 38.117.8 2.14 E269_12R 35.0 25.2 1.39 E269_13S 61.2 47.8 1.28 E269_14T 66.050.6 1.30 E269_15Y 57.6 44.7 1.29 E269_16H 50.7 38.5 1.32 E269_18N 59.244.8 1.32 E269_19Q 57.8 44.3 1.31 E269_20W 55.5 42.5 1.30 P271_04F 75.959.5 1.28 P271_15Y 70.7 52.3 1.35 P271_20W 77.3 58.1 1.33 Q295_01G 60.946.0 1.32 Q295_03V 91.2 75.7 1.20 Q295_04F 83.6 64.7 1.29 Q295_05P 87.467.5 1.30 Q295_11K 88.9 70.4 1.26 Q295_12R 83.0 65.2 1.27 Q295_13S 73.756.9 1.30 Q295_15Y 82.8 65.4 1.27 Q295_16H 81.3 62.7 1.30 Q295_18N 82.464.9 1.27 Q295_20W 60.0 44.4 1.35 Y296_01G 95.8 79.3 1.21 Y296_03V 96.279.3 1.21 Y296_05P 47.7 36.6 1.30 Y296_11K 94.3 72.8 1.30 Y296_12R 99.679.5 1.25 S298_01G 98.8 80.1 1.23 S298_02A 91.2 73.4 1.24 S298_03V 86.364.5 1.34 S298_04F 84.0 65.4 1.29 S298_05P 16.0 11.9 1.34 S298_07I 90.170.6 1.28 S298_09D 67.8 48.3 1.40

TABLE 20-3 REGION b IIa/IIb=>1.2 IIaR binding IIb binding IIaR/IIb NAMEHe/Con_2aR He/Con_2b He_2aR/2b S298_10E 65.1 47.6 1.37 S298_11K 95.161.3 1.55 S298_12R 85.3 57.8 1.48 S298_14T 98.5 77.9 1.26 S298_15Y 79.161.4 1.29 S298_16H 88.5 63.5 1.39 S298_18N 68.0 48.1 1.41 S298_19Q 95.073.3 1.30 S298_20W 55.4 41.1 1.35 Y300_05P 10.8 7.3 1.47 Y300_12R 55.141.2 1.34 Y300_18N 100.4 82.7 1.21 N325_02A 84.1 64.4 1.31 N325_05P 42.226.0 1.63 N325_11K 16.2 10.4 1.57 N325_12R 26.1 19.1 1.37 N325_16H 89.070.1 1.27 N325_19Q 92.1 69.1 1.33 P329_01G 55.0 41.7 1.32 P329_02A 58.940.5 1.45 P329_03V 53.8 38.5 1.40 P329_04F 44.7 32.2 1.39 P329_06M 42.729.1 1.47 P329_07I 49.6 34.6 1.44 P329_08L 42.8 29.1 1.47 P329_09D 67.853.5 1.27 P329_10E 64.8 52.7 1.23 P329_11K 30.2 16.9 1.79 P329_12R 29.317.6 1.66 P329_13S 61.5 41.0 1.50 P329_14T 57.3 37.9 1.51 P329_15Y 67.945.7 1.49 P329_16H 56.7 39.1 1.45 P329_18N 61.2 40.4 1.51 P329_19Q 41.130.7 1.34 A330_03V 74.9 52.4 1.43 A330_07I 85.6 67.1 1.27 A330_08L 88.870.5 1.26 A330_09D 70.9 55.6 1.27 A330_10E 86.6 70.1 1.23 A330_12R 103.584.8 1.22 A330_13S 60.8 41.3 1.47 A330_14T 95.5 77.9 1.23 A330_18N 81.163.7 1.27 P331_01G 61.0 45.4 1.34 I336_20W 30.3 20.3 1.50

The table shows a list of alterations that selectively enhance thebinding to FcγRIIa (R) as compared to FcγRIIb.

TABLE 21-1 REGION a IIaH > Con REGION b IIb < Con IIaH/Iib=>1.2 IIaHbinding IIb binding IIaH binding IIb binding IIaH/IIb NAME He/Con_2aHHe/Con_2b NAME He/Con_2aH He/Con_2b He_2aH/2b L234_04F 119.8 99.8L234_01G 75.6 50.7 1.49 L234_15Y 120.2 96.3 L234_02A 81.0 64.9 1.25G236_02A 139.2 96.5 L234_04F 119.8 99.8 1.20 G236_03V 119.4 35.3L234_05P 93.5 72.9 1.28 G236_04F 117.8 39.3 L234_11K 40.8 16.2 2.52G236_07I 115.2 31.0 L234_12R 47.7 18.9 2.53 G236_13S 134.9 96.7 L234_13S80.4 59.3 1.36 G236_14T 103.2 57.8 L234_14T 78.1 62.5 1.25 G236_15Y136.1 39.5 L234_15Y 120.2 96.3 1.25 G236_20W 146.7 58.2 L234_16H 87.359.4 1.47 H268_04F 111.0 89.6 L234_19Q 79.2 60.3 1.31 D270_10E 102.476.5 L235_01G 54.1 35.4 1.53 Q295_05P 103.3 67.5 L235_03V 85.3 66.4 1.28Q295_07I 102.3 84.3 L235_11K 22.7 6.3 3.61 Y296_04F 100.1 99.2 L235_12R30.3 9.6 3.16 Y296_09D 104.1 98.9 L235_13S 66.1 48.2 1.37 Y296_16H 103.792.2 L235_14T 71.5 46.8 1.53 Y296_18N 103.8 91.5 L235_18N 66.1 54.9 1.20Y300_06M 104.8 84.3 L235_19Q 62.0 42.6 1.45 Y300_07I 108.4 74.5 G236_02A139.2 96.5 1.44 Y300_08L 108.2 73.6 G236_03V 119.4 35.3 3.39 K326_12R100.6 89.2 G236_04F 117.8 39.3 3.00 S324_02A 102.4 78.3 G236_05P 63.519.0 3.34 S324_04F 101.4 89.1 G236_06M 85.7 45.5 1.88 S324_09D 105.785.8 G236_07I 115.2 31.0 3.72 S324_10E 102.8 77.4 G236_08L 75.8 26.82.83 S324_15Y 102.3 88.2 G236_12R 34.2 6.8 5.04 S324_16H 102.0 83.5G236_13S 134.9 96.7 1.39 S324_20W 103.1 87.5 G236_14T 103.2 57.8 1.79A330_11K 109.4 95.0 G236_15Y 136.1 39.5 3.45 A330_12R 107.7 84.8G236_16H 98.2 27.9 3.51 A330_16H 101.4 87.6 G236_19Q 89.1 46.6 1.91A330_19Q 102.2 90.8 G236_20W 146.7 58.2 2.52 I332_03V 104.7 80.7S239_11K 48.1 24.7 1.94 I332_13S 109.5 98.4 S239_12R 57.1 39.1 1.46I332_16H 109.2 93.0 S239_16H 59.4 49.4 1.20 I332_18N 110.0 89.9 D265_02A31.3 19.9 1.58 I332_19Q 108.0 94.3 D265_03V 4.6 3.2 1.42 E333_04F 101.694.6 D265_04F 4.1 3.2 1.28 E333_08L 101.5 95.0 D265_06M 13.5 10.0 1.35E333_09D 103.4 94.3 D265_07I 3.1 1.3 2.35 E333_13S 100.6 81.8 D265_11K27.8 14.9 1.87 E333_14T 100.4 86.1 D265_12R 28.0 15.0 1.87 T335_01G103.1 96.7 D265_13S 19.8 11.3 1.75 T335_02A 100.7 94.0 D265_14T 24.116.0 1.50 T335_05P 107.6 99.9 D265_15Y 3.7 0.6 6.07 T335_06M 102.7 99.4D265_16H 48.6 38.3 1.27 I336_03V 102.2 90.3 D265_18N 12.4 6.1 2.01I336_10E 101.5 89.8 D265_19Q 47.7 33.4 1.43 I336_18N 103.0 97.6 D265_20W3.2 1.1 2.86 V266_05P 0.7 0.4 1.91 H268_04F 111.0 89.6 1.24 H268_06M84.8 65.7 1.29 H268_08L 85.0 67.8 1.25 E269_01G 71.5 51.5 1.39

TABLE 21-2 REGION b IIaH/Iib=>1.2 IIaH binding IIb binding IIaH/IIb NAMEHe/Con_2aH He/Con_2b He_2aH/2b E269_02A 77.8 49.9 1.56 E269_03V 67.545.1 1.49 E269_04F 68.5 45.0 1.52 E269_06M 73.6 46.7 1.58 E269_07I 66.643.9 1.52 E269_08L 67.4 42.9 1.57 E269_11K 51.1 17.8 2.87 E269_12R 46.825.2 1.86 E269_13S 70.0 47.8 1.46 E269_14T 69.6 50.6 1.37 E269_15Y 70.444.7 1.57 E269_16H 59.0 38.5 1.53 E269_18N 64.4 44.8 1.44 E269_19Q 80.244.3 1.81 E269_20W 60.9 42.5 1.43 P271_15Y 64.6 52.3 1.24 P271_20W 77.458.1 1.33 D270_01G 52.5 30.5 1.72 D270_02A 76.4 48.3 1.58 D270_03V 61.839.5 1.56 D270_04F 75.4 53.1 1.42 D270_06M 74.4 48.2 1.54 D270_07I 64.041.5 1.54 D270_08L 75.8 53.6 1.41 D270_10E 102.4 76.5 1.34 D270_11K 44.020.3 2.17 D270_12R 39.5 18.3 2.16 D270_13S 71.2 44.2 1.61 D270_14T 75.357.8 1.30 D270_15Y 71.9 44.9 1.60 D270_16H 71.5 47.2 1.51 D270_18N 62.540.3 1.55 D270_19Q 85.0 48.0 1.77 D270_20W 60.2 34.7 1.74 Q295_03V 92.075.7 1.22 Q295_05P 103.3 67.5 1.53 Q295_07I 102.3 84.3 1.21 Q295_11K89.4 70.4 1.27 Q295_12R 84.1 65.2 1.29 Q295_13S 69.4 56.9 1.22 Q295_16H78.6 62.7 1.25 Q295_20W 55.6 44.4 1.25 S298_03V 85.1 64.5 1.32 S298_07I86.9 70.6 1.23 S298_10E 58.0 47.6 1.22 S298_14T 98.5 77.9 1.26 S298_16H82.1 63.5 1.29 S298_18N 61.6 48.1 1.28 S298_19Q 91.0 73.3 1.24 S298_20W49.9 41.1 1.21 Y300_01G 74.4 48.4 1.54 Y300_02A 93.5 69.2 1.35 Y300_03V98.0 73.9 1.33 Y300_06M 104.8 84.3 1.24 Y300_07I 108.4 74.5 1.45Y300_08L 108.2 73.6 1.47 Y300_11K 66.9 51.6 1.30

TABLE 21-3 REGION b IIaH/Iib=>1.2 IIaH binding IIb binding IIaH/IIb NAMEHe/Con_2aH He/Con_2b He_2aH/2b Y300_12R 55.4 41.2 1.34 Y300_13S 88.153.9 1.64 Y300_14T 94.1 55.6 1.69 N325_11K 16.1 10.4 1.55 N325_12R 29.919.1 1.56 S324_01G 98.8 81.1 1.22 S324_02A 102.4 78.3 1.31 S324_09D105.7 85.8 1.23 S324_10E 102.8 77.4 1.33 S324_11K 92.2 68.6 1.34S324_12R 96.5 63.1 1.53 S324_16H 102.0 83.5 1.22 S324_18N 97.8 81.1 1.21S324_19Q 96.0 66.8 1.44 A327_04F 84.4 63.7 1.32 A327_06M 75.5 59.5 1.27A327_12R 11.3 9.0 1.26 A327_19Q 67.3 54.2 1.24 L328_11K 32.2 7.5 4.27L328_12R 42.5 9.2 4.60 P329_01G 54.9 41.7 1.32 P329_02A 65.7 40.5 1.62P329_03V 60.4 38.5 1.57 P329_04F 44.5 32.2 1.38 P329_06M 43.7 29.1 1.50P329_07I 53.8 34.6 1.56 P329_08L 45.4 29.1 1.56 P329_11K 46.2 16.9 2.74P329_12R 46.4 17.6 2.63 P329_13S 67.3 41.0 1.64 P329_14T 62.4 37.9 1.65P329_15Y 68.7 45.7 1.50 P329_16H 66.1 39.1 1.69 P329_18N 66.2 40.4 1.64P329_19Q 38.7 30.7 1.26 A330_03V 79.7 52.4 1.52 A330_07I 87.3 67.1 1.30A330_08L 87.6 70.5 1.24 A330_12R 107.7 84.8 1.27 A330_13S 65.4 41.3 1.58A330_14T 94.5 77.9 1.21 A330_18N 81.6 63.7 1.28 I332_03V 104.7 80.7 1.30I332_12R 87.8 48.9 1.80 I332_18N 110.0 89.9 1.22 E333_01G 97.7 81.4 1.20E333_11K 98.1 80.6 1.22 E333_13S 100.6 81.8 1.23 E333_18N 96.2 76.1 1.26I336_19Q 73.7 58.2 1.27 I336_20W 30.9 20.3 1.52

The table shows a list of alterations that selectively enhance thebinding to FcγRIIa (H) as compared to FcγRII.

TABLE 22-1 REGION a IIIa > Con IIb < Con IIIa binding IIb binding NAMEHe/Con_3a He/Con_2b L234_04F 118.1 99.8 L234_07I 103.1 80.9 L234_15Y130.2 96.3 G236_15Y 120.8 39.5 G236_20W 135.4 58.2 D270_10E 123.4 76.5Q295_02A 105.6 79.7 Q295_10E 100.2 94.8 Q295_14T 114.7 66.8 S298_02A160.0 73.4 S298_03V 109.3 64.5 S298_14T 112.6 77.9 Y300_08L 104.9 73.6S324_01G 104.9 81.1 S324_02A 104.1 78.3 S324_09D 101.0 85.8 S324_14T103.1 88.4 S324_15Y 101.4 88.2 S324_16H 103.7 83.5 S324_20W 101.8 87.5A330_03V 100.9 52.4 A330_04F 147.1 92.8 A330_06M 141.6 80.5 A330_07I103.0 67.1 A330_08L 124.5 70.5 A330_15Y 125.0 95.0 A330_16H 104.2 87.6A330_20W 104.2 76.1 I332_01G 110.7 87.1 I332_13S 104.5 98.4 I332_19Q110.5 94.3 E333_02A 105.4 83.8 E333_03V 103.0 93.2 E333_04F 101.1 94.6E333_09D 112.1 94.3 E333_13S 102.7 81.8 E333_16H 103.0 83.3 E333_20W100.9 89.5 K334_01G 121.3 92.5 K334_09D 159.7 94.8 T335_05P 104.9 99.9T335_06M 101.5 99.4 I336_02A 111.0 84.9 I336_03V 112.2 90.3 I336_09D106.2 92.1 I336_10E 119.4 89.8 I336_13S 101.6 83.7 I336_14T 108.2 89.5I336_18N 104.0 97.6 S337_01G 102.2 92.0 S337_04F 101.4 97.8 REGION bIIIa/IIb => 1.2 IIb binding IIIa binding IIIa/Iib NAME He/Con_2bHe/Con_3a He_3a/2b L234_03V 73.1 91.4 1.25 L234_05P 72.9 88.1 1.21L234_07I 80.9 103.1 1.27 L234_11K 16.2 30.5 1.88 L234_12R 18.9 36.9 1.95L234_15Y 96.3 130.2 1.35 L234_16H 59.4 79.5 1.34 L234_19Q 60.3 79.8 1.32L235_01G 35.4 58.8 1.66 L235_03V 66.4 98.0 1.48 L235_05P 54.7 71.1 1.30L235_11K 6.3 38.1 6.06 L235_12R 9.6 41.2 4.29 L235_13S 48.2 69.6 1.44L235_14T 46.8 75.8 1.62 L235_18N 54.9 67.3 1.23 L235_19Q 42.6 70.5 1.65G236_03V 35.3 57.7 1.64 G236_04F 39.3 87.0 2.21 G236_05P 19.0 37.1 1.95G236_07I 31.0 59.7 1.93 G236_08L 26.8 32.5 1.21 G236_12R 6.8 12.5 1.84G236_15Y 39.5 120.8 3.06 G236_16H 27.9 43.1 1.54 G236_20W 58.2 135.42.33 H268_06M 65.7 80.3 1.22 E269_01G 51.5 62.2 1.21 E269_02A 49.9 68.71.38 E269_03V 45.1 56.6 1.25 E269_06M 46.7 58.5 1.25 E269_07I 43.9 55.81.27 E269_08L 42.9 52.1 1.21 E269_11K 17.8 44.0 2.47 E269_12R 25.2 43.21.72 E269_13S 47.8 62.7 1.31 E269_16H 38.5 51.2 1.33 E269_18N 44.8 57.81.29 E269_19Q 44.3 65.0 1.47 P271_20W 58.1 74.1 1.28 D270_01G 30.5 38.51.26 D270_03V 39.5 60.3 1.52 D270_06M 48.2 66.3 1.38 D270_07I 41.5 69.61.68 D270_08L 53.6 71.8 1.34 D270_10E 76.5 123.4 1.61 D270_11K 20.3 41.02.02 D270_12R 18.3 28.8 1.58 D270_13S 44.2 56.7 1.28 D270_15Y 44.9 54.51.22 D270_18N 40.3 56.5 1.40 D270_19Q 48.0 81.9 1.70 Q295_01G 46.0 73.01.59 Q295_02A 79.7 105.6 1.32 Q295_09D 60.8 81.5 1.34

TABLE 22-2 REGION b IIIa/IIb=>1.2 IIb binding IIIa binding IIIa/Iib NAMEHe/Con_2b He/Con_3a He_3a/2b Q295_13S 56.9 86.1 1.51 Q295_14T 66.8 114.71.72 Q295_16H 62.7 76.4 1.22 Q295_18N 64.9 81.6 1.26 Y296_20W 103.3135.0 1.31 S298_02A 73.4 160.0 2.18 S298_03V 64.5 109.3 1.69 S298_04F65.4 82.5 1.26 S298_07I 70.6 96.4 1.37 S298_09D 48.3 66.7 1.38 S298_14T77.9 112.6 1.45 S298_15Y 61.4 85.6 1.39 S298_16H 63.5 76.5 1.20 Y300_01G48.4 81.6 1.69 Y300_07I 74.5 95.1 1.28 Y300_08L 73.6 104.9 1.43 Y300_13S53.9 73.9 1.37 Y300_14T 55.6 85.4 1.53 S324_01G 81.1 104.9 1.29 S324_02A78.3 104.1 1.33 S324_10E 77.4 99.3 1.28 S324_11K 68.6 83.0 1.21 S324_12R63.1 90.3 1.43 S324_16H 83.5 103.7 1.24 S324_19Q 66.8 88.5 1.33 A327_11K12.5 31.7 2.53 A327_12R 9.0 19.3 2.15 L328_11K 7.5 33.5 4.46 L328_12R9.2 32.7 3.53 P329_02A 40.5 56.4 1.39 P329_11K 16.9 29.6 1.76 P329_12R17.6 26.7 1.52 A330_03V 52.4 100.9 1.93 A330_04F 92.8 147.1 1.59A330_06M 80.5 141.6 1.76 A330_07I 67.1 103.0 1.53 A330_08L 70.5 124.51.77 A330_10E 70.1 84.6 1.21 A330_15Y 95.0 125.0 1.32 A330_20W 76.1104.2 1.37 I332_01G 87.1 110.7 1.27 I332_09D 147.0 206.6 1.41 I332_10E142.4 232.7 1.63 I332_12R 48.9 75.9 1.55 E333_01G 81.4 98.2 1.21E333_02A 83.8 105.4 1.26 E333_13S 81.8 102.7 1.26 E333_16H 83.3 103.01.24 K334_01G 92.5 121.3 1.31 K334_02A 107.5 147.1 1.37 K334_04F 110.9147.5 1.33 K334_05P 105.1 133.7 1.27 K334_06M 109.1 144.5 1.33 K334_07I119.1 151.1 1.27 K334_08L 106.5 143.3 1.35 K334_09D 94.8 159.7 1.69

TABLE 22-3 REGION b IIIa/IIb=>1.2 IIb binding IIIa binding IIIa/Iib NAMEHe/Con_2b He/Con_3a He_3a/2b K334_10E 114.5 180.6 1.58 K334_13S 110.9145.3 1.31 K334_14T 115.4 141.4 1.23 K334_15Y 109.2 140.8 1.29 K334_19Q109.3 136.1 1.25 K334_20W 103.8 127.8 1.23 I336_02A 84.9 111.0 1.31I336_03V 90.3 112.2 1.24 I336_10E 89.8 119.4 1.33 I336_13S 83.7 101.61.21 I336_14T 89.5 108.2 1.21 I336_15Y 68.2 87.3 1.28 I336_19Q 58.2 92.51.59

The table shows a list of alterations that selectively enhance thebinding to FcγRIIIa as compared to FcγRIIb.

On the other hand, FcγRIIb, the sole inhibitory FcγR, plays an importantrole in autoimmune diseases and inflammatory diseases (J. Exp. Med.,203, 2157-2164, 2006; J. immunol., 178, 3272-3280, 2007). It has alsobeen shown that antibodies having an Fc domain with increasedFcγRIIb-binding activity can be effective in treating autoimmunediseases caused by B cells (Mol. Immunology 45, 3926-3933, 2008). In thecase of antibodies that aim at treating autoimmune diseases andinflammatory diseases, ADCC activity and ADCP activity via activatingFcγR can aggravate the pathological conditions. Thus, it is desirable toincrease the binding activity to inhibitory FcγR while reducing thebinding activity to activating FcγR as much as possible. Specifically,like the alterations present in Region c in FIG. 24, alterationsdesirably have the effect to increase the binding activity to inhibitoryFcγR as compared to the native antibody and to reduce the bindingactivity to activating FcγR. Such alterations can be said to bealterations that selectively enhance the binding activity to inhibitoryFcγR compared to activating FcγR. Furthermore, like the alterationspresent in Region d in FIG. 25, alterations desirably have the effect toincrease the ratio between the binding activity to inhibitory FcγR andthe binding activity to activating FcγR as compared to the nativeantibody. Such alterations can be said to be alterations thatselectively increase the binding activity to inhibitory FcγR as comparedto activating FcγR.

Based on FIGS. 26, 27, 28, and 29 on the assessment of the ratio betweenthe binding activity to inhibitory FcγR and activating FcγR for each ofthe above heterodimerized antibodies, for alterations shown in eachfigure, those present in Regions corresponding to c and d in FIGS. 24and 25 are summarized as a list in Table 23 (Tables 23-1 and 23-2),Table 24 (Tables 24-1 and 24-2), Table 25 (Tables 25-1 to 25-3), Table26 (Tables 26-1 to 26-4).

TABLE 23-1 REGION c Ia > Con IIb > Con Ia binding IIb binding NAMEHe/Con_1a He/Con_2b L234_09D 97.8 150.3 L234_20W 98.6 114.4 L235_04F97.1 118.5 L235_09D 84.8 106.2 L235_15Y 97.1 149.1 L235_20W 92.8 147.3G236_09D 96.6 167.0 G236_10E 93.7 110.2 G237_02A 90.3 123.6 G237_04F81.5 149.2 G237_06M 81.6 129.9 G237_08L 79.6 148.0 G237_09D 84.0 222.7G237_10E 75.1 114.1 G237_13S 92.2 110.8 G237_15Y 79.6 152.5 G237_18N82.5 180.2 G237_19Q 82.8 107.9 G237_20W 78.4 193.4 P238_09D 96.0 220.9P238_16H 96.0 115.2 P238_19Q 97.6 110.1 P238_20W 99.9 122.7 S239_03V95.9 108.4 S239_05P 85.3 101.5 S239_06M 99.0 100.9 S239_07I 97.8 106.8S239_08L 99.8 140.3 V266_06M 99.7 251.2 V266_07I 99.3 152.4 V266_08L98.1 236.4 S267_07I 100.0 192.4 A327_01G 99.0 115.5 N325_01G 97.1 100.7N325_03V 95.6 111.1 N325_04F 93.2 143.2 N325_06M 97.6 217.0 N325_07I94.8 171.4 N325_08L 97.5 196.6 N325_09D 99.9 124.3 N325_13S 96.8 177.2N325_20W 93.8 168.8 S324_06M 99.2 112.1 A327_18N 97.7 113.1 L328_02A98.5 144.5 L328_04F 97.9 218.8 L328_06M 98.7 141.6 L328_07I 99.4 160.3L328_13S 99.0 149.2 P331_03V 92.0 118.7 P331_04F 95.5 110.5 P331_06M93.6 101.6 P331_07I 93.4 114.5 P331_10E 95.0 115.5 P331_15Y 96.0 114.4REGION d Ia/Iib =< 0.8 Ia binding IIb binding Ia/IIb NAME He/Con_1aHe/Con_2b He_1a/2b L234_09D 97.8 150.3 0.65 L235_09D 84.8 106.2 0.80L235_15Y 97.1 149.1 0.65 L235_20W 92.8 147.3 0.63 G236_09D 96.6 167.00.58 G237_02A 90.3 123.6 0.73 G237_04F 81.5 149.2 0.55 G237_06M 81.6129.9 0.63 G237_08L 79.6 148.0 0.54 G237_09D 84.0 222.7 0.38 G237_10E75.1 114.1 0.66 G237_15Y 79.6 152.5 0.52 G237_18N 82.5 180.2 0.46G237_19Q 82.8 107.9 0.77 G237_20W 78.4 193.4 0.41 P238_04F 101.0 312.00.32 P238_06M 102.3 174.0 0.59 P238_08L 100.9 204.5 0.49 P238_09D 96.0220.9 0.43 P238_10E 101.5 232.0 0.44 P238_15Y 100.9 217.8 0.46 S239_01G101.3 144.2 0.70 S239_08L 99.8 140.3 0.71 S239_09D 101.0 205.8 0.49S239_10E 105.2 180.6 0.58 V266_06M 99.7 251.2 0.40 V266_07I 99.3 152.40.65 V266_08L 98.1 236.4 0.42 V266_12R 59.2 −1.1 −55.61 S267_02A 108.9243.3 0.45 S267_03V 106.0 192.3 0.55 S267_06M 105.9 209.5 0.51 S267_07I100.0 192.4 0.52 S267_09D 109.7 310.5 0.35 S267_10E 105.8 379.3 0.28S267_19Q 102.5 217.5 0.47 H268_01G 107.2 143.6 0.75 H268_02A 108.7 166.60.65 H268_09D 109.5 239.1 0.46 H268_10E 111.0 230.5 0.48 H268_13S 109.3159.6 0.68 H268_18N 107.9 156.3 0.69 P271_01G 104.8 193.3 0.54 K326_02A101.6 139.2 0.73 K326_03V 101.8 175.3 0.58 K326_04F 101.9 155.1 0.66K326_06M 102.5 164.8 0.62 K326_07I 100.6 198.8 0.51 K326_08L 100.2 176.60.57 K326_09D 103.4 192.9 0.54 K326_10E 103.7 200.1 0.52 K326_14T 104.1149.5 0.70 K326_15Y 105.9 159.4 0.66 K326_19Q 102.3 139.3 0.73 K326_20W103.2 148.5 0.69

TABLE 23-2 REGION c Ia > Con IIb > Con Ia binding IIb binding NAMEHe/Con_1a He/Con_2b P331_16H 92.4 116.0 P331_20W 94.5 111.2 I332_02A88.3 103.3 I332_04F 93.6 104.0 I332_06M 92.3 104.7 I332_09D 93.4 147.0I332_10E 96.3 142.4 I332_14T 91.8 106.4 I332_15Y 88.8 101.3 REGION dIa/Iib =< 0.8 Ia binding IIb binding Ia/IIb NAME He/Con_1a He/Con_2bHe_1a/2b N325_04F 93.2 143.2 0.65 N325_06M 97.6 217.0 0.45 N325_07I 94.8171.4 0.55 N325_08L 97.5 196.6 0.50 N325_13S 96.8 177.2 0.55 N325_20W93.8 168.8 0.56 A327_09D 102.8 171.7 0.60 A327_10E 103.4 140.5 0.74L328_02A 98.5 144.5 0.68 L328_04F 97.9 218.8 0.45 L328_06M 98.7 141.60.70 L328_07I 99.4 160.3 0.62 L328_09D 101.6 198.8 0.51 L328_10E 101.2172.2 0.59 L328_13S 99.0 149.2 0.66 L328_14T 100.4 141.3 0.71 L328_15Y101.6 192.9 0.53 L328_20W 102.8 234.6 0.44 A330_05P 101.4 136.7 0.74P331_03V 92.0 118.7 0.78 P331_16H 92.4 116.0 0.80 I332_09D 93.4 147.00.64 I332_10E 96.3 142.4 0.68

The table shows a list of alterations that selectively enhance thebinding to FcγRIIb as compared to FcγRIa.

TABLE 24-1 REGION c IIaR < Con IIb > Con IIaR binding IIb binding NAMEHe/Con_2aR He/Con_2b S239_03V 99.9 108.4 S267_08L 72.1 108.7 S267_14T81.9 104.4 Y300_19Q 99.7 108.3 A327_01G 98.2 115.5 A327_07I 98.0 126.7A327_18N 88.9 113.1 L328_19Q 89.3 104.9 I332_02A 97.6 103.3 REGION dIIaR/Iib =< 0.8 IIaR binding IIb binding IIaR/IIb NAME He/Con_2aRHe/Con_2b He_2aR/2b G236_09D 106.1 167.0 0.64 G237_09D 155.6 222.7 0.70G237_20W 138.3 193.4 0.71 P238_04F 174.4 312.0 0.56 P238_08L 139.7 204.50.68 P238_09D 148.2 220.9 0.67 P238_10E 151.3 232.0 0.65 P238_15Y 156.2217.8 0.72 S239_09D 136.6 205.8 0.66 S239_10E 134.9 180.6 0.75 V266_04F44.4 57.6 0.77 V266_05P −2.9 0.4 −8.33 V266_06M 149.8 251.2 0.60V266_07I 120.3 152.4 0.79 V266_08L 141.7 236.4 0.60 V266_11K −5.6 1.0−5.55 S267_02A 155.3 243.3 0.64 S267_03V 116.5 192.3 0.61 S267_04F 37.848.0 0.79 S267_06M 122.5 209.5 0.58 S267_07I 110.8 192.4 0.58 S267_08L72.1 108.7 0.66 S267_09D 173.0 310.5 0.56 S267_10E 174.6 379.3 0.46S267_11K 3.6 12.2 0.30 S267_12R 3.6 11.3 0.32 S267_14T 81.9 104.4 0.78S267_16H 50.6 65.9 0.77 S267_19Q 135.2 217.5 0.62 H268_02A 133.2 166.60.80 H268_09D 149.3 239.1 0.62 H268_10E 147.7 230.5 0.64 H268_13S 127.3159.6 0.80 P271_01G 144.0 193.3 0.75 D270_05P 7.5 13.9 0.54 Y300_10E106.6 133.8 0.80 K326_07I 147.0 198.8 0.74 K326_08L 136.8 176.6 0.77K326_09D 148.0 192.9 0.77 K326_10E 147.8 200.1 0.74 N325_06M 152.3 217.00.70 N325_07I 133.2 171.4 0.78 N325_08L 143.8 196.6 0.73 N325_20W 133.8168.8 0.79 A327_07I 98.0 126.7 0.77 A327_09D 123.8 171.7 0.72 A327_10E110.1 140.5 0.78 A327_12R 6.0 9.0 0.67 A327_18N 88.9 113.1 0.79 L328_04F137.9 218.8 0.63 L328_07I 124.2 160.3 0.78 L328_09D 119.0 198.8 0.60L328_10E 105.4 172.2 0.61 L328_11K 2.5 7.5 0.33 L328_12R 5.0 9.2 0.54

TABLE 24-2 REGION d IIaR/Iib=<0.8 IIaR binding IIb binding IIaR/IIb NAMEHe/Con_2aR He/Con_2b He_2aR/2b L328_13S 116.6 149.2 0.78 L328_15Y 135.5192.9 0.70 L328_18N 59.4 80.8 0.73 L328_20W 155.1 234.6 0.66 I332_11K43.1 70.9 0.61

The table shows a list of alterations that selectively enhance thebinding to FcγRIIb as compared to FcγRIIa(R).

TABLE 25-1 REGION c IIaH < Con IIb > Con IIaH binding IIb binding NAMEHe/Con_2aH He/Con_2b L234_09D 89.9 150.3 L234_10E 88.3 117.6 L235_09D75.8 106.2 G236_09D 89.8 167.0 G237_02A 34.0 123.6 G237_04F 41.4 149.2G237_06M 17.8 129.9 G237_08L 13.9 148.0 G237_09D 11.1 222.7 G237_10E15.1 114.1 G237_13S 29.8 110.8 G237_15Y 25.4 152.5 G237_18N 15.2 180.2G237_19Q 26.5 107.9 G237_20W 31.6 193.4 P238_03V 89.0 120.1 P238_04F65.4 312.0 P238_06M 47.6 174.0 P238_08L 84.6 204.5 P238_09D 87.5 220.9P238_10E 77.1 232.0 P238_15Y 59.8 217.8 P238_16H 45.9 115.2 P238_19Q56.4 110.1 P238_20W 59.5 122.7 S239_01G 97.0 144.2 S239_03V 91.0 108.4S239_05P 33.3 101.5 S239_06M 98.6 100.9 S239_07I 91.5 106.8 S239_18N94.2 119.1 V266_06M 83.9 251.2 S267_01G 71.3 104.6 S267_03V 50.8 192.3S267_06M 34.3 209.5 S267_07I 34.7 192.4 S267_08L 29.0 108.7 S267_14T63.3 104.4 S267_19Q 61.3 217.5 H268_01G 99.2 143.6 H268_03V 99.2 113.9H268_05P 81.8 116.1 H268_20W 95.8 111.4 P271_08L 86.7 102.7 P271_09D70.1 120.1 P271_10E 63.1 109.2 Y296_20W 97.6 103.3 S298_06M 98.1 101.8S298_08L 85.0 108.5 Y300_10E 97.2 133.8 Y300_19Q 96.1 108.3 K326_03V97.0 175.3 K326_04F 97.0 155.1 K326_05P 98.8 125.6 K326_08L 92.7 176.6REGION d IIaH/Iib =< 0.8 IIaH binding IIb binding IIaH/IIb NAMEHe/Con_2aH He/Con_2b He_2aH/2b L234_09D 89.9 150.3 0.60 L234_10E 88.3117.6 0.75 L235_09D 75.8 106.2 0.71 L235_20W 117.7 147.3 0.80 G236_09D89.8 167.0 0.54 G237_02A 34.0 123.6 0.27 G237_03V 39.4 61.4 0.64G237_04F 41.4 149.2 0.28 G237_05P 9.5 24.6 0.39 G237_06M 17.8 129.9 0.14G237_08L 13.9 148.0 0.09 G237_09D 11.1 222.7 0.05 G237_10E 15.1 114.10.13 G237_11K 5.8 9.0 0.64 G237_12R 7.5 9.8 0.76 G237_13S 29.8 110.80.27 G237_14T 31.8 54.8 0.58 G237_15Y 25.4 152.5 0.17 G237_16H 16.4 55.20.30 G237_18N 15.2 180.2 0.08 G237_19Q 26.5 107.9 0.25 G237_20W 31.6193.4 0.16 P238_01G 37.3 56.0 0.66 P238_03V 89.0 120.1 0.74 P238_04F65.4 312.0 0.21 P238_06M 47.6 174.0 0.27 P238_07I 57.6 80.0 0.72P238_08L 84.6 204.5 0.41 P238_09D 87.5 220.9 0.40 P238_10E 77.1 232.00.33 P238_13S 50.9 70.4 0.72 P238_14T 37.7 50.7 0.74 P238_15Y 59.8 217.80.27 P238_16H 45.9 115.2 0.40 P238_18N 53.5 73.2 0.73 P238_19Q 56.4110.1 0.51 P238_20W 59.5 122.7 0.49 S239_01G 97.0 144.2 0.67 S239_05P33.3 101.5 0.33 S239_08L 104.8 140.3 0.75 S239_09D 114.4 205.8 0.56S239_10E 112.0 180.6 0.62 S239_18N 94.2 119.1 0.79 S239_20W 35.2 45.10.78 V266_01G 36.7 51.5 0.71 V266_04F 25.3 57.6 0.44 V266_06M 83.9 251.20.33 V266_07I 105.5 152.4 0.69 V266_08L 104.5 236.4 0.44 V266_09D 6.410.1 0.63 V266_10E 10.1 15.2 0.66 V266_12R 1.1 −1.1 −1.05 V266_13S 27.054.4 0.50 V266_14T 43.3 82.3 0.53 V266_15Y 18.1 49.4 0.37

TABLE 25-2 REGION c IIaH < Con IIb > Con IIaH binding IIb binding NAMEHe/Con_2aH He/Con_2b K326_15Y 98.3 159.4 K326_16H 97.9 118.7 K326_20W83.3 148.5 A327_01G 97.2 115.5 N325_01G 56.8 100.7 N325_03V 45.2 111.1N325_04F 33.4 143.2 N325_06M 48.2 217.0 N325_07I 38.1 171.4 N325_08L52.0 196.6 N325_09D 53.1 124.3 N325_13S 79.7 177.2 N325_20W 47.2 168.8A327_07I 58.4 126.7 A327_10E 91.3 140.5 A327_18N 66.8 113.1 L328_03V97.8 125.7 L328_04F 71.7 218.8 L328_07I 93.9 160.3 L328_09D 58.5 198.8L328_10E 55.5 172.2 L328_15Y 90.0 192.9 L328_19Q 86.8 104.9 A330_01G99.0 103.5 A330_05P 85.7 136.7 P331_03V 82.5 118.7 P331_04F 84.7 110.5P331_06M 85.1 101.6 P331_07I 77.4 114.5 P331_10E 87.1 115.5 P331_15Y86.0 114.4 P331_16H 84.5 116.0 P331_20W 82.8 111.2 K334_02A 97.0 107.5K334_05P 97.6 105.1 K334_10E 98.2 114.5 K334_12R 99.7 111.2 K334_18N99.7 114.8 K334_20W 97.6 103.8 S337_15Y 99.4 100.2 S337_18N 99.8 103.3REGION d IIaH/Iib =< 0.8 IIaH binding IIb binding IIaH/IIb NAMEHe/Con_2aH He/Con_2b He_2aH/2b V266_16H 21.2 57.2 0.37 V266_19Q 46.489.2 0.52 V266_20W 12.0 71.9 0.17 S267_01G 71.3 104.6 0.68 S267_02A119.8 243.3 0.49 S267_03V 50.8 192.3 0.26 S267_04F 22.7 48.0 0.47S267_05P 25.1 31.5 0.80 S267_06M 34.3 209.5 0.16 S267_07I 34.7 192.40.18 S267_08L 29.0 108.7 0.27 S267_09D 105.4 310.5 0.34 S267_10E 102.3379.3 0.27 S267_11K 9.3 12.2 0.76 S267_12R 8.5 11.3 0.76 S267_14T 63.3104.4 0.61 S267_15Y 19.6 37.9 0.52 S267_16H 29.8 65.9 0.45 S267_19Q 61.3217.5 0.28 S267_20W 37.9 57.2 0.66 H268_01G 99.2 143.6 0.69 H268_02A113.8 166.6 0.68 H268_05P 81.8 116.1 0.70 H268_09D 132.6 239.1 0.55H268_10E 124.3 230.5 0.54 H268_13S 112.4 159.6 0.70 H268_18N 112.1 156.30.72 H268_19Q 102.4 132.8 0.77 P271_01G 107.0 193.3 0.55 P271_09D 70.1120.1 0.58 P271_10E 63.1 109.2 0.58 P271_14T 60.9 95.4 0.64 S298_08L85.0 108.5 0.78 Y300_10E 97.2 133.8 0.73 K326_02A 105.6 139.2 0.76K326_03V 97.0 175.3 0.55 K326_04F 97.0 155.1 0.63 K326_05P 98.8 125.60.79 K326_06M 103.3 164.8 0.63 K326_07I 100.1 198.8 0.50 K326_08L 92.7176.6 0.52 K326_09D 110.3 192.9 0.57 K326_10E 101.8 200.1 0.51 K326_14T114.5 149.5 0.77 K326_15Y 98.3 159.4 0.62 K326_19Q 101.4 139.3 0.73K326_20W 83.3 148.5 0.56 N325_01G 56.8 100.7 0.56 N325_02A 47.6 64.40.74 N325_03V 45.2 111.1 0.41 N325_04F 33.4 143.2 0.23 N325_05P 20.726.0 0.80 N325_06M 48.2 217.0 0.22 N325_07I 38.1 171.4 0.22 N325_08L52.0 196.6 0.26 N325_09D 53.1 124.3 0.43 N325_10E 52.5 95.9 0.55

TABLE 25-3 REGION d IIaH/Iib=<0.8 IIaH binding IIb binding IIaH/IIb NAMEHe/Con_2aH He/Con_2b He_2aH/2b N325_13S 79.7 177.2 0.45 N325_14T 62.791.6 0.68 N325_15Y 41.2 99.5 0.41 N325_16H 48.0 70.1 0.68 N325_20W 47.2168.8 0.28 S324_05P 55.5 94.7 0.59 A327_03V 51.0 94.7 0.54 A327_07I 58.4126.7 0.46 A327_08L 46.9 63.1 0.74 A327_09D 104.7 171.7 0.61 A327_10E91.3 140.5 0.65 A327_14T 45.6 89.4 0.51 A327_18N 66.8 113.1 0.59L328_03V 97.8 125.7 0.78 L328_04F 71.7 218.8 0.33 L328_05P 45.4 65.10.70 L328_06M 103.5 141.6 0.73 L328_07I 93.9 160.3 0.59 L328_09D 58.5198.8 0.29 L328_10E 55.5 172.2 0.32 L328_15Y 90.0 192.9 0.47 L328_20W112.6 234.6 0.48 A330_05P 85.7 136.7 0.63 P331_03V 82.5 118.7 0.70P331_04F 84.7 110.5 0.77 P331_07I 77.4 114.5 0.68 P331_10E 87.1 115.50.75 P331_15Y 86.0 114.4 0.75 P331_16H 84.5 116.0 0.73 P331_20W 82.8111.2 0.75 I332_10E 108.3 142.4 0.76

The table shows a list of alterations that selectively enhance thebinding to FcγRIIb as compared to FcγRIIa(H).

TABLE 26-1 REGION c IIIa < Con IIb > Con IIIa binding IIb binding NAMEHe/Con_3a He/Con_2b L235_04F 79.1 118.5 L235_09D 91.6 106.2 L235_15Y80.6 149.1 L235_20W 76.4 147.3 G236_09D 71.2 167.0 G236_10E 70.7 110.2G237_02A 48.8 123.6 G237_04F 25.3 149.2 G237_06M 31.9 129.9 G237_08L24.9 148.0 G237_09D 17.7 222.7 G237_10E 25.4 114.1 G237_13S 38.4 110.8G237_15Y 22.1 152.5 G237_18N 20.3 180.2 G237_19Q 43.3 107.9 G237_20W19.7 193.4 P238_03V 43.8 120.1 P238_04F 46.7 312.0 P238_06M 30.5 174.0P238_08L 75.5 204.5 P238_15Y 64.5 217.8 P238_16H 30.7 115.2 P238_19Q55.8 110.1 P238_20W 49.7 122.7 S239_01G 76.2 144.2 S239_03V 76.6 108.4S239_05P 14.9 101.5 S239_06M 78.2 100.9 S239_07I 79.3 106.8 S239_08L95.3 140.3 V266_06M 85.8 251.2 S267_01G 53.8 104.6 S267_03V 30.8 192.3S267_06M 66.0 209.5 S267_07I 25.2 192.4 S267_08L 19.5 108.7 S267_10E91.9 379.3 S267_14T 45.6 104.4 S267_19Q 65.0 217.5 H268_01G 96.7 143.6H268_03V 92.3 113.9 H268_05P 76.4 116.1 H268_15Y 97.5 106.3 H268_20W82.8 111.4 P271_08L 71.0 102.7 P271_09D 93.1 120.1 P271_10E 93.6 109.2S298_06M 92.6 101.8 S298_08L 73.5 108.5 Y300_19Q 96.3 108.3 K326_16H96.1 118.7 K326_20W 84.8 148.5 A327_01G 72.4 115.5 N325_01G 28.0 100.7REGION d IIIa/Iib =< 0.8 IIb binding IIIa binding IIIa/Iib NAMEHe/Con_2b He/Con_3a He_3a/2b L234_09D 150.3 116.8 0.78 L235_04F 118.579.1 0.67 L235_15Y 149.1 80.6 0.54 L235_16H 76.9 59.1 0.77 L235_20W147.3 76.4 0.52 G236_09D 167.0 71.2 0.43 G236_10E 110.2 70.7 0.64G236_11K 49.8 18.6 0.37 G236_13S 96.7 64.8 0.67 G236_18N 96.4 37.8 0.39G237_02A 123.6 48.8 0.39 G237_04F 149.2 25.3 0.17 G237_05P 24.6 10.90.44 G237_06M 129.9 31.9 0.25 G237_08L 148.0 24.9 0.17 G237_09D 222.717.7 0.08 G237_10E 114.1 25.4 0.22 G237_12R 9.8 7.4 0.76 G237_13S 110.838.4 0.35 G237_15Y 152.5 22.1 0.14 G237_16H 55.2 17.2 0.31 G237_18N180.2 20.3 0.11 G237_19Q 107.9 43.3 0.40 G237_20W 193.4 19.7 0.10P238_01G 56.0 33.0 0.59 P238_02A 57.4 31.4 0.55 P238_03V 120.1 43.8 0.36P238_04F 312.0 46.7 0.15 P238_06M 174.0 30.5 0.18 P238_07I 80.0 27.80.35 P238_08L 204.5 75.5 0.37 P238_09D 220.9 105.8 0.48 P238_10E 232.0104.7 0.45 P238_11K 18.1 10.4 0.57 P238_12R 51.4 12.2 0.24 P238_13S 70.444.9 0.64 P238_14T 50.7 28.0 0.55 P238_15Y 217.8 64.5 0.30 P238_16H115.2 30.7 0.27 P238_18N 73.2 40.4 0.55 P238_19Q 110.1 55.8 0.51P238_20W 122.7 49.7 0.40 S239_01G 144.2 76.2 0.53 S239_03V 108.4 76.60.71 S239_04F 44.1 25.3 0.57 S239_05P 101.5 14.9 0.15 S239_06M 100.978.2 0.78 S239_07I 106.8 79.3 0.74 S239_08L 140.3 95.3 0.68 S239_11K24.7 11.9 0.48 S239_12R 39.1 17.0 0.44 S239_15Y 34.9 25.6 0.73 S239_16H49.4 29.2 0.59 S239_19Q 86.1 56.9 0.66 D265_01G 16.1 4.5 0.28

TABLE 26-2 REGION c IIIa < Con IIb > Con IIIa binding IIb binding NAMEHe/Con_3a He/Con_2b N325_03V 30.3 111.1 N325_04F 20.7 143.2 N325_06M31.4 217.0 N325_07I 28.7 171.4 N325_08L 33.3 196.6 N325_09D 48.0 124.3N325_13S 65.1 177.2 N325_20W 32.8 168.8 A327_07I 42.4 126.7 A327_09D83.7 171.7 A327_10E 68.6 140.5 A327_18N 61.1 113.1 L328_02A 79.1 144.5L328_03V 78.7 125.7 L328_04F 79.3 218.8 L328_06M 78.6 141.6 L328_07I84.0 160.3 L328_09D 47.8 198.8 L328_10E 53.4 172.2 L328_13S 71.7 149.2L328_14T 75.3 141.3 L328_15Y 68.3 192.9 L328_19Q 81.1 104.9 L328_20W37.4 234.6 A330_01G 89.1 103.5 P331_03V 73.2 118.7 P331_04F 77.3 110.5P331_06M 74.8 101.6 P331_07I 66.0 114.5 P331_10E 75.1 115.5 P331_15Y81.9 114.4 P331_16H 80.2 116.0 P331_20W 78.6 111.2 I332_04F 97.6 104.0K334_12R 90.8 111.2 T335_04F 96.0 102.9 T335_18N 98.8 101.6 S337_07I94.8 104.0 S337_08L 96.9 100.2 REGION d IIIa/Iib =< 0.8 IIb binding IIIabinding IIIa/Iib NAME He/Con_2b He/Con_3a He_3a/2b D265_02A 19.9 7.40.37 D265_03V 3.2 0.6 0.19 D265_04F 3.2 0.0 0.00 D265_06M 10.0 2.6 0.26D265_07I 1.3 −0.8 −0.61 D265_08L 4.6 0.0 0.00 D265_10E 38.2 20.1 0.53D265_11K 14.9 2.5 0.17 D265_12R 15.0 3.2 0.21 D265_13S 11.3 3.9 0.35D265_14T 16.0 4.3 0.27 D265_15Y 0.6 −1.7 −2.86 D265_16H 38.3 13.6 0.35D265_18N 6.1 4.6 0.74 D265_19Q 33.4 8.5 0.25 D265_20W 1.1 −0.5 −0.48V266_01G 51.5 24.0 0.47 V266_02A 81.0 57.4 0.71 V266_04F 57.6 34.7 0.60V266_05P 0.4 −0.7 −1.98 V266_06M 251.2 85.8 0.34 V266_07I 152.4 114.10.75 V266_08L 236.4 118.0 0.50 V266_09D 10.1 2.7 0.27 V266_10E 15.2 4.10.27 V266_11K 1.0 −0.7 −0.70 V266_13S 54.4 28.1 0.52 V266_14T 82.3 39.40.48 V266_15Y 49.4 14.5 0.29 V266_16H 57.2 21.3 0.37 V266_18N 69.7 30.40.44 V266_19Q 89.2 34.7 0.39 V266_20W 71.9 9.0 0.13 S267_01G 104.6 53.80.51 S267_02A 243.3 150.4 0.62 S267_03V 192.3 30.8 0.16 S267_04F 48.011.0 0.23 S267_05P 31.5 15.7 0.50 S267_06M 209.5 66.0 0.31 S267_07I192.4 25.2 0.13 S267_08L 108.7 19.5 0.18 S267_09D 310.5 180.8 0.58S267_10E 379.3 91.9 0.24 S267_14T 104.4 45.6 0.44 S267_15Y 37.9 11.80.31 S267_16H 65.9 17.9 0.27 S267_19Q 217.5 65.0 0.30 S267_20W 57.2 21.50.37 H268_01G 143.6 96.7 0.67 H268_02A 166.6 122.2 0.73 H268_05P 116.176.4 0.66 H268_13S 159.6 121.9 0.76 H268_18N 156.3 104.1 0.67 H268_20W111.4 82.8 0.74 P271_01G 193.3 113.2 0.59 P271_08L 102.7 71.0 0.69

TABLE 26-3 REGION d IIIa/Iib=<0.8 IIb binding IIIa binding IIIa/Iib NAMEHe/Con_2b He/Con_3a He_3a/2b P271_09D 120.1 93.1 0.77 P271_14T 95.4 56.40.59 Y296_01G 79.3 54.5 0.69 Y296_11K 72.8 56.7 0.78 Y296_13S 86.3 61.80.72 Y296_14T 88.2 64.5 0.73 Y296_16H 92.2 73.4 0.80 Y296_18N 91.5 72.10.79 S298_05P 11.9 8.2 0.69 S298_08L 108.5 73.5 0.68 Y300_05P 7.3 2.80.38 Y300_10E 133.8 104.2 0.78 K326_03V 175.3 122.1 0.70 K326_04F 155.1102.9 0.66 K326_06M 164.8 114.8 0.70 K326_07I 198.8 138.6 0.70 K326_08L176.6 114.2 0.65 K326_09D 192.9 133.6 0.69 K326_10E 200.1 124.5 0.62K326_15Y 159.4 112.3 0.70 K326_19Q 139.3 106.3 0.76 K326_20W 148.5 84.80.57 A327_01G 115.5 72.4 0.63 N325_01G 100.7 28.0 0.28 N325_02A 64.430.5 0.47 N325_03V 111.1 30.3 0.27 N325_04F 143.2 20.7 0.14 N325_05P26.0 10.2 0.39 N325_06M 217.0 31.4 0.14 N325_07I 171.4 28.7 0.17N325_08L 196.6 33.3 0.17 N325_09D 124.3 48.0 0.39 N325_10E 95.9 38.20.40 N325_11K 10.4 5.2 0.50 N325_13S 177.2 65.1 0.37 N325_14T 91.6 39.40.43 N325_15Y 99.5 29.5 0.30 N325_19Q 69.1 31.6 0.46 N325_20W 168.8 32.80.19 S324_05P 94.7 65.5 0.69 A327_03V 94.7 45.8 0.48 A327_04F 63.7 46.20.72 A327_07I 126.7 42.4 0.33 A327_08L 63.1 42.0 0.67 A327_09D 171.783.7 0.49 A327_10E 140.5 68.6 0.49 A327_14T 89.4 41.4 0.46 A327_15Y 60.846.8 0.77 A327_16H 63.5 43.7 0.69 A327_18N 113.1 61.1 0.54 A327_20W 78.851.2 0.65 L328_01G 80.2 44.9 0.56 L328_02A 144.5 79.1 0.55 L328_03V125.7 78.7 0.63 L328_04F 218.8 79.3 0.36 L328_05P 65.1 40.5 0.62

TABLE 26-4 REGION d IIIa/Iib=<0.8 IIb binding IIIa binding IIIa/Iib NAMEHe/Con_2b He/Con_3a He_3a/2b L328_06M 141.6 78.6 0.56 L328_07I 160.384.0 0.52 L328_09D 198.8 47.8 0.24 L328_10E 172.2 53.4 0.31 L328_13S149.2 71.7 0.48 L328_14T 141.3 75.3 0.53 L328_15Y 192.9 68.3 0.35L328_16H 80.6 54.3 0.67 L328_18N 80.8 49.7 0.61 L328_19Q 104.9 81.1 0.77L328_20W 234.6 37.4 0.16 P329_20W 45.1 31.7 0.70 P331_03V 118.7 73.20.62 P331_04F 110.5 77.3 0.70 P331_06M 101.6 74.8 0.74 P331_07I 114.566.0 0.58 P331_08L 95.7 72.1 0.75 P331_10E 115.5 75.1 0.65 P331_11K 84.767.5 0.80 P331_14T 99.8 74.1 0.74 P331_15Y 114.4 81.9 0.72 P331_16H116.0 80.2 0.69 P331_18N 99.2 73.4 0.74 P331_20W 111.2 78.6 0.71I332_11K 70.9 45.1 0.64

The table shows a list of alterations that selectively enhance thebinding to FcγRIIb as compared to FcγRIIIa.

[Example 9] Assessment of Heterodimerized Antibodies for PhysicochemicalStability

When antibodies are developed as pharmaceuticals, antibodies areexpected to have high physicochemical stability. For example, as to anantibody alteration where the above-described S239D, A330L, and I332Eare introduced into both antibody chains, it has been reported that theantibody Fc domain becomes thermodynamically unstable due to theintroduced alteration (Molecular Immunology, 45, 1872-1882, 2008), andsuch reduced thermal stability makes it difficult to develop antibodiesas pharmaceuticals. In order to increase the usefulness of antibodies aspharmaceuticals and to make the development simpler, it is alsoimportant to increase the FcγR-binding activity and maintain thephysicochemical stability. In the case of homodimerized antibodies,alterations are introduced into both H chains, and this means that anantibody molecule introduced with a single type of alteration has thealteration at two sites. In the case of heterodimerized antibodies, onthe other hand, it is possible to choose whether an alteration isintroduced into either H chain, and thus, even when introducing a singletype of alteration, it is introduced at only one site per an antibodymolecule. As discussed in Example 7, depending on the type ofalteration, as to the effect to increase the FcγRIIIa-binding activity,it is sometimes sufficient to introduce an alteration into one H chain.If an alteration has the effect to reduce the physicochemical stabilityof an antibody, one can confer the antibody with the effect to increasethe FcγRIIIa-binding activity by introducing the alteration into onlyone H chain while minimizing the physicochemical destabilization of theantibody.

To assess this hypothesis, the present inventors assessed which residueof S239D, A330L, and I332E actually contributed to the destabilizationof the CH2 domain. Expression vectors inserted with the following wereprepared according to the method described in Reference Example 1:GpH7-B3-06-09D (SEQ ID NO: 37), GpH7-B3-20-08L (SEQ ID NO: 38), andGpH7-B3-22-10E (SEQ ID NO: 39) resulting from the introduction of thealterations S239D, A330L, and I332E, respectively, into GpH7-B3. UsingGpL16-k0 as the L chain, antibodies of interest were expressed andprepared according to the method described in Reference Example 1.Furthermore, as a control, an antibody of interest was expressed andprepared using GpL16-k0 and GpH7-B3 which does not contain thealteration. The respective antibodies were compared for the Tm of theCH2 domain by thermal shift assay according to the method described inReference Example 5 (Table 27). Hereinafter, unless otherwise specified,Tm means to the Tm of CH2 domain.

TABLE 27 MUTATION Tm ΔTm SAMPLE H SITE (° C.) (° C.) GpH7-B3/GpL16-k0 B3— 68 0 (SEQ ID NO: 4, 5) GpH7-B3-06-09D/GpL16-k0 B3-6-09D S239D 65 −3(SEQ ID NO: 37, 5) GpH7-B3-20-08L/GpL16-k0 B3-20-08L A330L 67 −1 (SEQ IDNO: 38, 5) GpH7-B3-22-10E/GpL16-k0 B3-22-10E I332E 60 −8 (SEQ ID NO: 39,5)

The column “SAMPLE” indicates antibody names; the column “H” indicatesnames of the H chain constant region of each antibody; the column“MUTATION SITE” indicates mutations that are different in comparisonwith GpH7-B3/GpL16-k0 (“-”: when there is no particular mutation); thecolumn “Tm” indicates the Tm of each antibody; and the column “ΔTm”indicates the difference in the Tm between each antibody andGpH7-B3/GpL16-k0. The SEQ ID NOs are also shown for the amino acidsequences of the H chain and L chain of each antibody.

When the homodimerized antibody GpH7-B3-06-09D/GpL16-k0 introduced withS239D, the homodimerized antibody GpH7-B3-20-08L/GpL16-k0 introducedwith A330L, and the homodimerized antibody GpH7-B3-22-10E/GpL16-k0introduced with I332E were compared to GpH7-B3/GpL16-k0, their Tm wasdecreased by 3° C., 1° C., and 8° C., respectively. This resultdemonstrates that, of the three alterations, I332E had the greatesteffect to reduce the Tm of CH2, thus I332E is thought to also contributeto the decrease of Tm of an antibody introduced with the group of thealterations S239D, A330L, and I332E.

The side chain of I332E is surrounded by hydrophobic amino acids such asV240, V323, and L328. In an antibody introduced with I332E, thehydrophobic interaction with surrounding residues is abolished due tothe substitution of hydrophobic Ile with hydrophilic Glu, and this isthought to contribute to the destabilization of the Fc domain.Meanwhile, as discussed in Example 7, the interaction of I332E withFcγRIIIa occurs exclusively in either H chain. For this reason, it wasthought that when I332 is kept unsubstituted in the other H chain thatis not involved in the interaction with FcγRIIIa, the thermodynamicstability can be maintained while conferring the effect to enhance theFcγRIIIa binding. Then, the present inventors introduced I332E into onlyone H chain, and assessed whether the Tm is elevated as compared to whenI332E is introduced into both H chains. Expression vectors inserted withGpH7-A44 (SEQ ID NO: 49) and GpH7-B80 (SEQ ID NO: 50) resulting from theintroduction of I332E into GpH7-A5 and GpH7-B3, respectively, wereconstructed. They were combined with GpH7-B3, GpH7-A5, and GpL16-k0 toexpress and prepare the following antibodies according to the methoddescribed in Reference Example 1: the heterodimerized antibodiesGpH7-A5/GpH7-B80/GpL16-k0 and GpH7-A44/GpH7-B3/GpL16-k0 in which onlyone of the H chains was introduced with I332E, and the homodimerizedantibody GpH7-A44/GpH7-B80/GpL16-k0 in which both of the H chains wereintroduced with I332E. GpH7-A5/GpH7-B3/GpL16-k0 was prepared as acontrol. Each antibody was assessed for the FcγRIIIa-binding activityaccording to the method described in Reference Example 2. In addition,the antibodies were compared for the Tm of CH2 domain by thermal shiftassay according to the method described in Reference Example 5 (Tables28 and 29).

TABLE 28 MUTATION MUTATION KD KD SAMPLE H1 SITE H2 SITE (M) ratioGpH7-A5/GpH7-B3/GpL16-k0 A5 — B3 — 1.6E−06 1.0 (SEQ ID NO: 3, 4, 5)GpH7-A5/GpH7-B80/GpL16-k0 A5 — B80 I332E 4.7E−07 3.4 (SEQ ID NO: 3, 50,5) GpH7-A44/GpH7-B3/GpL16-k0 A44 I332E B3 — 3.7E−07 4.4 (SEQ ID NO: 49,4, 5) GpH7-A44/GpH7-B80/GpL16-k0 A44 I332E B80 I332E 2.3E−07 7.1 (SEQ IDNO: 49, 50, 5)

The column “SAMPLE” indicates antibody names; the columns “H1” and “H2”indicate names of the H chain constant region of each antibody; and thecolumn “MUTATION SITE” indicates mutations that are different incomparison with GpH7-A5/GpH7-B3/GpL16-k0 (“-”: when there is noparticular mutation). A value obtained by dividing KD ofGpH7-A5/GpH7-B3/GpL16-k0 for FcγRIIIa by the KD of each antibody wasdefined as “KD ratio”. The SEQ ID NOs are also shown for the amino acidsequences of the H chain and L chain of each antibody.

TABLE 29 MUTATION MUTATION Tm ΔTm SAMPLE H1 SITE H2 SITE (° C.) (° C.)GpH7-A5/GpH7-B3/GpL16-k0 A5 — B3 — 68 0 (SEQ ID NO: 3, 4, 5)GpH7-A5/GpH7-B80/GpL16-k0 A5 — B80 I332E 64 −4 (SEQ ID NO: 3, 50, 5)GpH7-A44/GpH7-B3/GpL16-k0 A44 I332E B3 — 64 −4 (SEQ ID NO: 49, 4, 5)GpH7-A44/GpH7-B80/GpL16-k0 A44 I332E B80 I332E 58 −10 (SEQ ID NO: 49,50, 5)

The table shows the Tm of CH2 of antibodies with I332E substitution inone H chain, and antibodies with I332E substitution in both H chains.

The column “SAMPLE” indicates antibody names; the columns “H1” and “H2”indicate names of the H chain constant region of each antibody; thecolumn “MUTATION SITE” indicates mutations that are different incomparison with GpH7-A5/GpH7-B3/GpL16-k0 (“-”: when there is noparticular mutation); the column “Tm” indicates the Tm of each antibody;and the column “ΔTm” indicates the difference in the Tm between eachantibody and GpH7-B3/GpL16-k0. The SEQ ID NOs are also shown for theamino acid sequences of the H chain and L chain of each antibody.

The result shown in Table 28 demonstrates that the FcγRIIIa-bindingactivity of the heterodimerized antibody GpH7-A5/GpH7-B80/GpL16-k0 inwhich only one of the H chains was introduced with I332E was increasedby about 3 times that of GpH7-A5/GpH7-B3/GpL16-k0, while the activity ofGpH7-A44/GpH7-B3/GpL16-k0 was increased by about 4 times that ofGpH7-A5/GpH7-B3/GpL16-k0. From this, it was thought that the effect ofI332E to increase the FcγRIIIa-binding activity is not significantlyaltered regardless of when I332E is introduced into GpH7-A5 or it isintroduced into GpH7-B3. Meanwhile, the FcγRIIIa-binding activity ofGpH7-A44/GpH7-B80/GpL16-k0 in which both of the H chains were introducedwith I332E was increased by about 7 times that ofGpH7-A5/GpH7-B3/GpL16-k0. These findings revealed that I332E has aneffect to sufficiently increase the FcγRIIIa-binding activity even ifintroduced into only one H chain, if not introduced into both H chains,as considered based on the three-dimensional structure.

Furthermore, the result shown in Table 29 revealed that the Tm of theheterodimerized antibodies GpH7-A5/GpH7-B80/GpL16-k0 andGpH7-A44/GpH7-B3/GpL16-k0 in which I332E was introduced into only one Hchain, were both decreased by 4° C. compared to the Tm ofGpH7-A5/GpH7-B3/GpL16-k0 which is their parental Fc molecule. Thus, itwas thought that there is no difference in the influence of I332E onantibody Tm regardless of when I332E is introduced into GpH7-A5 orGpH7-B3. Meanwhile, the Tm of the homodimerized antibodyGpH7-A44/GpH7-B80/GpL16-k0 in which both of the H chains were introducedwith I332E was decreased by 10° C. compared to that ofGpH7-A5/GpH7-B3/GpL16-k0. The Tm of the heterodimerized antibody inwhich only one of the H chains was introduced with I332E was maintainedto be 6° C. higher than that of the homodimerized antibody in which bothof the H chains were introduced with I332E. This result demonstratesthat the decrease in the Tm can be suppressed by using a heterodimerizedantibody in which only one, not both, of the H chains is introduced withI332E. This shows that the heterodimerized antibody technology is usefulto maintain the antibody physicochemical stability.

The alteration I332E is superior in terms of the effect to enhance theFcγRIIIa binding; however, if conventional homodimerized antibodies areused with it, their thermodynamic stability is significantly reduced,and this is problematic when antibodies are used as pharmaceuticals.However, the results shown in Tables 28 and 29 demonstrate that the useof a heterodimerized antibody enables exploiting the effect of I332E toenhance the FcγRIIIa while maintaining the physicochemical antibodystability. From this finding, it is thought that heterodimerizedantibodies are an excellent technology for more finely adjusting theFcγR-binding activity and the physicochemical stability of antibodies.

GpH7-TA7/GpH7-B78/GpL16-k0 has S239D, A330L, and I332E in only one Hchain, and thus possibly retains high Tm as compared toGpH7-A57/GpH7-B78/GpL16-k0 in which both of the H chains have S239D,A330L, and I332E. Thus, the heterodimerized and homodimerized antibodiesresulting from the combination of the group of the alterations L234Y,G236W, and S298A with the group of the alterations S239D, A330L, andI332E described in Example 7 were assayed for Tm according to the methoddescribed in Reference Example 5.

To assess this, the heterodimerized antibody GpH7-TA7/GpH7-B78/GpL16-k0in which one of the H chains was introduced with the group of thealterations L234Y, G236W, and S298A and the other H chain was introducedwith the group of the alterations S239D, A330L, and I332E; theheterodimerized antibody GpH7-TA7/GpH7-B3/GpL16-k0 in which only one ofthe H chains was introduced with the group of the alterations L234Y,G236W, and S298A; the homodimerized antibody GpH7-TA7/GpH7-TA45/GpL16-k0in which both of the H chains were introduced with the group of thealterations L234Y, G236W, and S298A; the heterodimerized antibodyGpH7-A5/GpH7-B78/GpL16-k0 in which only one of the H chains wasintroduced with S239D, A330L, and I332E, and the homodimerized antibodyGpH7-A57/GpH7-B78/GpL16-k0 in which both of the H chains were introducedwith S239D, A330L, and I332E, were prepared according to the methoddescribed in Reference Example 1. The influence of the combination ofL234Y, G236W, and S298A with S239D, A330L, and I332E on the Tm wasassessed by comparing the Tm of CH2 domain of the respective antibodiesby thermal shift assay according to the method described in ReferenceExample 5 (Table 30).

TABLE 30 MUTATION MUTATION Tm ΔTm Sample H1 SITE H2 SITE (° C.) (° C.)GpH7-G1d/GpL16-k0 G1d — — G1d — — 69 1 (SEQ ID NO: 2, 5)GpH7-A5/GpH7-B3/GpL16-k0 A5 D356K/H435R — B3 K439E — 68 0 (SEQ ID NO: 3,4, 5) GpH7-TA7/GpH7-B3/GpL16-k0 TA7 D356K/H435R L234Y/G236W/S298A B3K439E — 68 0 (SEQ ID NO: 31, 4, 5) GpH7-A5/GpH7-B78/GpL16-k0 A5D356K/H435R — B78 K439E S239D/A330L/I332E 60 −8 (SEQ ID NO: 3, 41, 5)GpH7-A57/GpH7-B78/GpL16-k0 A57 D356K/H435R S239D/A330L/I332E B78 K439ES239D/A330L/I332E 48 −20 (SEQ ID NO: 40, 41, 5)GpH7-TA7/GpH7-TA45/GpL16-k0 TA7 D356K/H435R L234Y/G236W/S298A TA45 K439EL234Y/G236W/S298A 68 0 (SEQ ID NO: 31, 32, 5) GpH7-TA7/GpH7-B78/GpL16-k0TA7 D356K/H435R L234Y/G236W/S298A B78 K439E S239D/A330L/I332E 59 −9 (SEQID NO: 31, 41, 5)

The column “Sample” indicates antibody names; the columns “H1” and “H2”indicate names of the H chain constant region of each antibody; thecolumn “MUTATION SITE” indicates mutations that are different incomparison with GpH7-G1d/GpL16-k0 (“-”: when there is no particularmutation); the column “Tm” indicates the Tm of each antibody; and thecolumn “ΔTm” indicates the difference in the Tm between each antibodyand GpH7-A5/GpH7-B3/GpL16-k0. The SEQ ID NOs are also shown for theamino acid sequences of the H chain and L chain of each antibody.

GpH7-A5/GpH7-B3/GpL16-k0 introduced with alterations D356K/H435R andK439E to increase the efficiency of heterodimerized antibody formationwas compared to GpH7-G1d/GpL16-k0 which is a native IgG1. The Tm of CH2was decreased by 1° C.

Homodimerized antibodies of the prior-art technology were assessed forthe effect of each alteration group. The Tm of the homodimerizedantibody GpH7-A57/GpH7-B78/GpL16-k0 in which both of the H chains wereintroduced with S239D, A330L, and I332E was decreased by 20° C. ascompared to GpH7-A5/GpH7-B3/GpL16-k0. Meanwhile, a decrease in the Tmwas not observed for the homodimerized antibodyGpH7-TA7/GpH7-TA45/GpL16-k0 in which both of the H chains wereintroduced with the group of the alterations L234Y, G236W, and S298A.Thus, it was thought that the group of the alterations L234Y, G236W, andS298A does not have the effect to decrease the Tm of homodimerizedantibodies.

Heterodimerized antibodies in which only one of the H chains wasintroduced with each alteration group were assessed for the effect ofeach alteration group. The Tm of the heterodimerized antibodyGpH7-A5/GpH7-B78/GpL16-k0 in which one of the H chains was introducedwith S239D, A330L, and I332E was decreased by 8° C. as compared toGpH7-A5/GpH7-B3/GpL16-k0. Meanwhile, a decrease in Tm was not observedfor the heterodimerized antibody GpH7-TA7/GpH7-B3/GpL16-k0 in which oneof the H chains was introduced with L234Y, G236W, and S298A. From thesefindings, it was thought that the group of the alterations L234Y, G236W,and S298A does not have the effect to decrease the Tm of heterodimerizedantibodies either.

The Tm of GpH7-A57/GpH7-B78/GpL16-k0 in which both of the H chains wereintroduced with S239D, A330L, and I332E was decreased by 21° C. ascompared to the native IgG1. The Tm of GpH7-A5/GpH7-B78/GpL16-k0 inwhich only one of the H chains has the alterations S239D, A330L, andI332E was 60° C., and it retained Tm 10° C. or more higher than that ofthe homodimerized antibody. As shown in Table 13 of Example 7, theFcγRIIIa binding of the homodimerized antibody with S239D, A330L, andI332E was increased by about 9 times that of the heterodimerizedantibody with S239D, A330L, and I332E. S239D, A330L, and I332E, whenintroduced into both H chains, strongly enhance the FcγRIIIa binding,but significantly reduce the Tm.

Furthermore, the Tm of GpH7-TA7/GpH7-TA45/GpL16-k0 in which both of theH chains were introduced with L234Y, G236W, and S298A was only decreasedby 1° C. as compared to the native antibody. The decrease of Tm wasthought to be due to the influence of D356K/H435R and K439E used toconstruct the heterodimerized antibody as discussed above, rather thandue to L234Y, G236W, and S298A. This is also shown by the fact that theTm of GpH7-TA7/GpH7-B3/GpL16-k0 in which one of the H chains wasintroduced with L234Y, G236W, and S298A was also only decreased by 1° C.

Finally, the Tm of GpH7-TA7/GpH7-B78/GpL16-k0 in which one of the Hchains has L234Y, G236W, and S298A and the other H chain has S239D,A330L, and I332E was decreased by 10° C. compared to the nativeantibody, and was almost equivalent to that of GpH7-A5/GpH7-B78/GpL16-k0in which one of the H chains was introduced with S239D, A330L, andI332E. However, as shown in Table 13 of Example 7, the FcγRIIIa bindingof GpH7-TA7/GpH7-B78/GpL16-k0 is enhanced by 10 times or more comparedto that of GpH7-A5/GpH7-B78/GpL16-k0.

That is, it was demonstrated that, by using the heterodimerized antibodyGpH7-TA7/GpH7-B78/GpL16-k0 in which one of the H chains was introducedwith L234Y, G236W, and S298A and the other was introduced with S239D,A330L, and I332E, the FcγRIIIa binding can be enhanced, and also, the Tmcan be increased by 10° C. or more, as compared to the homodimerizedantibody GpH7-A57/GpH7-B78/GpL16-k0 with S239D, A330L, and I332E.

Then, the above described samples subjected to Tm measurement werefurther assessed for the thermodynamic stability by the heat acceleratedstability study described in Reference Example 6 (at 40° C. for two orfour weeks) (FIG. 30).

When GpH7-A5/GpH7-B3/GpL16-k0 was compared to the native antibodyGpH7-G1d/GpH7-G1d/GpL16-k0, the monomer contents of the former andlatter were decreased by 1.27% and 1.86% after four weeks, respectively,and there was no significant difference. Thus, it was thought that thealterations D356K/H435R and K439E used to construct the heterodimerizedantibody have almost no influence on the change in the monomer contentin heat accelerated stability study.

Regarding GpH7-A57/GpH7-B78/GpL16-k0 in which both of the H chains wereintroduced with S239D, A330L, and I332E, the monomer content wasdecreased by about 16% after four weeks. Meanwhile, the monomer contentof GpH7-A5/GpH7-B78/GpL16-k0 in which only one of the H chains has thealterations S239D, A330L, and I332E was decreased by 9.63% after fourweeks. Thus, it was demonstrated that the effect of more stablymaintaining the monomer content was achieved by using heterodimerizedantibodies in which only one of the H chains was introduced with S239D,A330L, and I332E.

Furthermore, the monomer content of GpH7-TA7/GpH7-TA45/GpL16-k0 in whichboth of the H chains were introduced with L234Y, G236W, and S298A andGpH7-TA7/GpH7-B3/GpL16-k0 in which one of the H chains was introducedwith L234Y, G236W, and S298A was only decreased by 1.78% and 1.42% afterfour weeks, respectively. There was no clear difference in the monomercontent change as compared to the native antibody. Thus, it was thoughtthat L234Y, G236W, and S298A do not affect the monomer content in heataccelerated stability study even when they are introduced into one Hchain or both H chains.

Finally, the monomer content of GpH7-TA7/GpH7-B78/GpL16-k0 in which oneof the H chains has L234Y, G236W, and S298A and the other H chain hasS239D, A330L, and I332E was decreased by 2.47% after four weeks. Themonomer content was only slightly reduced compared to 1.86% for thenative antibody. Thus, it was demonstrated that, by using theheterodimerized antibody GpH7-TA7/GpH7-B78/GpL16-k0 in which one of theH chains has L234Y, G236W, and S298A and the other H chain has S239D,A330L, and I332E, the FcγRIIIa binding can be enhanced, and also, theeffect of retaining the monomer content at a high level in heataccelerated stability study can be achieved as compared to thehomodimerized antibody GpH7-A57/GpH7-B78/GpL16-k0 with S239D, A330L, andI332E.

Thus, it was demonstrated that, when compared to conventionalhomodimerized antibodies, the technology of heterodimerized antibodiesis not only capable of enhancing the FcγR binding but also improves thestability, and thereby increases the value of antibodies aspharmaceuticals more than homodimerized antibodies.

[Example 10] Search for Alterations that Improve FcγR Binding but do notReduce the Stability

As described in Example 9, the FcγR-binding activity is increased byintroducing alterations in the H chain. However, such alterations canreduce the physicochemical stability of CH2, i.e., reduce the Tm.However, as described in Example 9, such properties are unfavorable, inparticular when antibodies are used as pharmaceuticals. As described inExample 9, it is useful to use the heterodimerized antibody in whichonly one of the H chains has been introduced with alterations to enhancethe FcγR-binding activity while suppressing the destabilization of CH2.Specifically, with respect to alterations that decrease the Tm ofhomodimerized antibodies even though they are observed to enhance theFcγR-binding activity of conventional homodimerized antibodies, such asthose corresponding to Regions ii and iii shown in FIG. 21, theirheterodimerization allows the Tm to be higher than that of thehomodimerized antibodies while increasing the FcγR-binding activity ascompared to the native antibody.

To find such alterations, homodimerized antibodies in Regions ii and iiishown in FIG. 21 were assayed for Tm according to the method describedin Reference Example 5. A list of alterations that reduce the Tm ascompared to the native antibody is shown in Tables 31 to 35.

Table 31 (Tables 31-1 to 31-3) shows data for Ia where Tm is 68° C. orless for Region ii or iii; Table 32 (Tables 32-1 and 32-2) shows datafor IIaR where Tm is 68° C. or less for Region ii or iii; Table 33(Tables 33-1 and 33-2) shows data for IIaH where Tm is 68° C. or lessfor Region ii or iii; Table 34 (Tables 34-1 and 34-2) shows data for IIbwhere Tm is 68° C. or less for Region ii or iii; Table 35 (Tables 35-1and 35-2) shows data for IIIa where Tm is 68° C. or less for Region iior iii.

Antibodies that have improved FcγR binding as compared to the nativeantibody and whose stability is not significantly reduced can beproduced by using a heterodimerized antibody in which only one of the Hchains is introduced with these alterations.

TABLE 31-1 NAME Tm Ho/Con_1a He/Con_1a A327_09D 65.68 101.6 102.8A330_08L 66.88 106.1 106.9 A330_09D 65.88 108.0 108.6 A330_14T 67.88104.8 105.1 E269_04F 67 102.4 103.1 E269_06M 68 101.3 103.6 E269_08L67.72 101.1 103.6 E269_15Y 67.92 100.2 106.4 E333_01G 61.48 100.4 102.7E333_13S 65.88 100.6 103.1 G236_20W 64.8 104.4 105.2 I336_01G 53.28104.3 108.7 I336_02A 61.8 103.1 106.0 I336_03V 67.2 104.8 105.2 I336_04F56.28 103.9 105.5 I336_06M 61.68 104.0 108.2 I336_10E 53.8 107.2 107.8I336_18N 62 103.6 107.8 K326_14T 64.08 102.9 104.1 K326_15Y 65.72 101.2105.9 K326_16H 66.68 100.9 103.4 K334_10E 63.6 106.9 109.8 P271_02A 67102.5 103.5 P271_03V 66.2 102.0 102.3 P271_04F 66.68 100.3 101.8P271_06M 66.68 102.0 102.5 P271_07I 66.12 101.9 102.5 P271_11K 65.2101.1 102.8 P271_12R 65.12 101.2 102.4 P271_13S 67.12 100.7 102.4P271_20W 67.32 101.5 102.6 Q295_02A 66.52 104.1 104.3 Q295_03V 67.8101.6 104.1 Q295_04F 67.8 100.3 102.2 Q295_05P 64.32 101.1 103.4Q295_07I 66.48 101.2 103.0 Q295_13S 65.52 101.4 102.0 Q295_14T 65.92102.5 102.7 Q295_18N 65.88 100.8 102.4 S239_01G 64.92 100.7 101.3S239_09D 65 100.8 101.0 S267_01G 67.2 102.2 103.2 S267_02A 66.72 108.5108.9 S267_03V 66 102.4 106.0 S267_09D 65.4 105.8 109.7 S298_03V 65106.2 109.7 S298_07I 64.32 103.3 107.0 S298_08L 65.52 104.2 106.7S298_10E 67 106.1 108.2 S298_14T 66.08 106.5 107.1 S298_15Y 65.68 103.1107.2 S298_16H 65.12 106.8 108.8 S298_18N 67.2 103.9 108.1 S298_19Q66.52 104.7 109.4 S324_02A 66.32 102.6 102.7 S324_03V 66.28 103.0 103.6S324_08L 67.4 102.7 103.2 S324_14T 67.12 112.4 120.9 S324_19Q 67.2 110.7112.3 S324_20W 58.2 111.5 114.3 S337_01G 64.92 102.3 106.6 S337_02A 67103.1 105.8 S337_03V 65.32 105.1 106.7

TABLE 31-2 NAME Tm Ho/Con_1a He/Con_1a S337_04F 65.88 104.7 106.2S337_06M 66.4 102.1 106.9 S337_07I 63.32 105.3 105.3 S337_08L 62.6 105.3105.3 S337_09D 56.52 105.5 105.7 S337_10E 62.08 104.2 106.4 S337_15Y65.48 103.6 103.8 S337_19Q 66.88 104.6 104.6 S337_20W 64.4 104.3 104.9T335_04F 64.6 104.8 105.0 T335_05P 57.12 106.2 106.4 T335_06M 66.2 106.0106.7 T335_07I 67 105.1 109.1 T335_08L 64.88 105.4 114.6 T335_09D 61.2106.8 107.5 T335_10E 62.92 107.8 108.8 T335_11K 67 102.8 110.8 T335_12R67 106.0 112.8 T335_15Y 64.72 106.3 107.2 T335_16H 64.28 104.5 105.7T335_18N 63.8 102.2 105.8 T335_19Q 64.92 104.6 105.0 T335_20W 65 104.2106.2 Y296_06M 65.68 103.8 104.0 Y296_11K 67 102.0 104.7 Y296_12R 67.32102.9 106.3 Y296_16H 67.32 105.3 105.8 Y296_20W 66.72 106.2 110.8Y300_02A 66.2 109.8 112.2 Y300_03V 63.6 112.4 117.0 Y300_06M 61.92 111.0112.2 Y300_08L 60.6 109.3 113.1 Y300_11K 66 101.2 106.9 Y300_13S 64.52110.3 111.1 Y300_14T 63.72 109.6 111.7 Y300_19Q 63 110.1 112.5 Y300_20W67.52 110.5 112.4 A330_01G 67.0 106.4 105.7 A330_04F 67.3 106.9 106.1A330_10E 67.2 108.1 107.4 A330_19Q 67.4 105.9 105.2 A330_20W 67.9 108.2107.6 D265_10E 61.4 103.4 101.4 H268_01G 67.5 108.0 107.2 H268_09D 67.1110.5 109.5 H268_10E 67.6 111.6 111.0 H268_13S 67.8 109.7 109.3 H268_18N66.8 108.4 107.9 I336_07I 67.9 106.1 104.4 I336_08L 64.8 109.4 107.4I336_09D 53.5 107.7 103.6 I336_11K 54.7 106.8 105.4 I336_12R 55.7 108.7102.0 I336_13S 57.4 109.5 102.5 I336_14T 64.1 111.2 104.5

TABLE 31-3 NAME Tm Ho/Con_1a He/Con_1a I336_15Y 57.9 109.7 102.9I336_16H 53.4 114.0 101.4 K326_03V 66.5 103.3 101.8 K326_04F 65.3 102.3101.9 K326_06M 66.6 103.7 102.5 K326_07I 66.1 101.6 100.6 K326_08L 67.1101.0 100.2 K326_09D 68.0 105.5 103.4 K326_10E 68.0 105.4 103.8 K326_19Q67.9 104.6 102.3 K326_20W 65.5 104.4 103.2 K334_01G 64.2 105.6 103.5K334_02A 65.8 106.0 105.9 K334_03V 67.0 105.3 104.7 K334_04F 65.9 106.1105.3 K334_05P 62.0 106.2 104.3 K334_07I 67.3 106.8 104.3 K334_08L 66.2107.6 104.7 K334_09D 65.6 107.6 106.5 K334_13S 66.1 105.8 104.9 K334_14T65.7 105.5 104.8 K334_15Y 65.9 106.6 104.7 K334_16H 62.8 105.9 104.9K334_18N 65.4 107.6 104.6 K334_19Q 67.1 106.1 106.1 L328_15Y 65.5 104.5101.6 P271_01G 68.0 105.5 104.8 P271_08L 66.4 103.4 102.2 P271_10E 67.9103.3 102.8 P271_18N 66.2 103.4 103.2 P271_19Q 66.5 103.6 103.1 Q295_10E64.9 104.7 103.1 Q295_12R 63.9 101.4 100.4 S239_10E 66.0 105.5 105.2S267_06M 66.4 106.4 105.9 S267_10E 64.1 106.9 105.8 S267_14T 67.8 107.4101.5 S267_18N 66.6 106.9 102.4 S267_19Q 66.8 103.6 102.5 S298_09D 67.4106.5 105.9 S324_09D 66.2 105.5 103.5 S324_10E 66.9 106.3 104.9 S337_11K67.3 104.7 104.0 S337_14T 67.3 106.3 105.1 S337_16H 66.6 104.8 104.7S337_18N 64.1 106.2 105.3 T335_01G 63.2 105.8 104.4 T335_02A 65.8 105.2103.3 T335_13S 67.6 105.1 104.8 T335_14T 68.0 107.0 106.5 Y296_02A 67.0103.4 102.9 Y296_03V 66.7 104.4 104.0 Y296_07I 65.9 105.9 104.3 Y296_08L66.9 104.9 103.9 Y300_07I 60.5 109.1 107.8 Y300_16H 63.7 111.1 110.9

TABLE 32-1 NAME Tm Ho/Con_2aR He/Con_2aR A327_09D 65.7 120.0 123.8A327_10E 65.4 103.7 110.2 A330_01G 67.0 105.9 108.9 A330_12R 67.9 100.8103.5 G237_04F 66.8 112.8 126.2 G237_20W 67.6 107.1 138.3 H268_01G 67.5122.0 124.2 I332_06M 65.0 105.4 108.8 I336_07I 67.9 105.8 108.3 I336_08L64.8 105.4 110.2 K326_03V 66.5 138.9 140.7 K326_04F 65.3 132.7 135.1K326_07I 66.1 133.2 147.0 K326_08L 67.1 130.7 136.8 K326_15Y 65.7 133.8136.4 K326_20W 65.5 119.9 130.8 K334_02A 65.8 100.1 104.9 K334_04F 65.9109.5 110.4 K334_05P 62.0 101.5 103.1 K334_08L 66.2 105.1 106.2 K334_10E63.6 105.7 105.7 K334_13S 66.1 105.6 109.1 K334_19Q 67.1 108.1 109.1L235_20W 67.9 114.4 130.9 L328_13S 64.5 107.9 116.6 L328_14T 64.9 118.2118.9 P238_03V 66.8 115.8 120.4 P238_04F 66.4 146.3 174.4 P271_08L 66.4102.5 103.8 P331_02A 66.0 102.2 106.0 P331_04F 63.0 106.3 110.6 P331_15Y63.2 105.2 112.4 P331_16H 63.0 100.2 114.0 P331_20W 61.8 101.9 109.8S239_08L 67.6 105.6 121.5 S239_10E 66.0 121.7 134.9 S239_18N 67.5 103.0110.7 S267_03V 66.0 114.7 116.5 S267_06M 66.4 102.3 122.5 S267_09D 65.4167.1 173.0 S267_19Q 66.8 110.3 135.2 S298_08L 65.5 101.4 104.9 S324_06M66.1 117.9 123.1 S337_02A 67.0 101.5 105.0 S337_03V 65.3 102.0 106.1S337_06M 66.4 100.9 105.2 S337_07I 63.3 102.8 108.9 S337_09D 56.5 113.5114.8 S337_10E 62.1 110.0 111.6 S337_15Y 65.5 100.4 105.4 S337_16H 66.6106.6 108.6 S337_18N 64.1 103.1 106.6 S337_19Q 66.9 101.8 105.6 S337_20W64.4 104.7 110.5 T335_02A 65.8 102.3 102.4 T335_05P 57.1 103.6 103.6T335_07I 67.0 106.7 109.2 T335_10E 62.9 108.7 111.3 T335_13S 67.6 101.6109.6 T335_14T 68.0 106.0 109.8

TABLE 32-2 NAME Tm Ho/Con_2aR He/Con_2aR T335_15Y 64.7 107.1 111.4T335_16H 64.3 102.5 108.2 T335_18N 63.8 100.4 106.3 T335_19Q 64.9 102.1106.0 T335_20W 65.0 101.2 108.7 V266_06M 64.8 145.6 149.9 Y296_20W 66.7106.9 109.8 G236_09D 66.2 107.1 106.1 G236_10E 65.9 139.8 124.9 H268_09D67.1 157.2 149.3 H268_10E 67.6 153.6 147.7 H268_13S 67.8 135.2 127.3H268_18N 66.8 134.4 128.4 I332_04F 61.9 108.6 105.0 I332_09D 56.2 126.8118.9 I332_10E 60.1 123.3 114.1 I332_15Y 59.4 111.1 109.6 K326_06M 66.6142.9 137.9 K326_09D 68.0 158.1 148.0 K326_10E 68.0 155.5 147.8 K326_14T64.1 140.8 134.0 K326_16H 66.7 122.7 120.0 K326_19Q 67.9 137.4 129.2K334_03V 67.0 123.3 117.2 K334_07I 67.3 120.9 113.8 K334_14T 65.7 117.2111.9 K334_15Y 65.9 112.5 108.4 K334_16H 62.8 110.7 109.4 K334_18N 65.4113.0 111.7 L328_03V 64.9 116.3 116.2 L328_04F 67.2 144.8 137.9 L328_06M67.7 124.2 115.4 L328_07I 66.0 140.3 124.2 L328_15Y 65.5 156.6 135.5P238_08L 67.4 148.0 139.7 P271_01G 68.0 150.0 144.0 S239_01G 64.9 132.0127.9 S239_09D 65.0 141.9 136.6 S267_01G 67.2 116.1 112.8 S267_02A 66.7159.9 155.3 S267_07I 65.7 113.2 110.8 S337_14T 67.3 110.5 110.0 T335_04F64.6 108.4 107.6 T335_06M 66.2 106.7 104.7 T335_08L 64.9 108.1 108.0T335_09D 61.2 113.4 109.9 Y300_04F 62.8 109.3 107.8

TABLE 33-1 NAME Tm Ho/Con_2aH He/Con_2aH G236_03V 67.9 110.7 119.4G236_07I 65.6 101.0 115.2 H268_18N 66.8 110.1 112.1 I336_06M 61.7 100.3106.1 I336_07I 67.9 103.6 104.8 I336_08L 64.8 100.9 105.5 K326_06M 66.6101.1 103.3 K326_10E 68.0 101.0 101.8 K326_19Q 67.9 100.5 101.4 K334_04F65.9 103.3 105.7 K334_08L 66.2 103.4 104.2 K334_16H 62.8 101.4 103.9L235_20W 67.9 111.8 117.7 L328_06M 67.7 100.2 103.5 L328_14T 64.9 110.3116.2 S239_09D 65.0 112.4 114.4 S267_02A 66.7 119.5 119.8 S324_02A 66.3101.5 102.5 S337_09D 56.5 106.9 107.1 S337_10E 62.1 101.3 102.4 T335_01G63.2 100.9 103.1 T335_07I 67.0 105.4 107.7 T335_10E 62.9 105.2 107.6T335_13S 67.6 101.4 106.1 T335_14T 68.0 104.3 107.9 T335_15Y 64.7 101.5106.3 T335_16H 64.3 100.4 105.3 T335_19Q 64.9 102.8 103.1 A330_12R 67.9110.2 107.7 G236_10E 65.9 121.9 111.2 H268_09D 67.1 135.6 132.6 H268_10E67.6 126.4 124.3 H268_13S 67.8 113.9 112.4 I332_02A 63.2 119.0 111.2I332_03V 67.8 105.6 104.7 I332_04F 61.9 111.2 104.1 I332_06M 65.0 113.5109.7 I332_09D 56.2 130.4 117.7 I332_10E 60.1 117.4 108.3 I332_13S 60.9120.8 109.5 I332_14T 63.1 125.1 114.9 I332_15Y 59.4 125.7 114.9 I332_16H60.6 118.6 109.2 I332_18N 58.0 118.5 110.0 I332_19Q 62.8 114.0 108.0K326_09D 68.0 113.3 110.3 K326_14T 64.1 118.4 114.5 K334_03V 67.0 111.1109.4 K334_07[ 67.3 115.5 110.1 K334_14T 65.7 105.1 103.2 K334_15Y 65.9110.4 106.0 L328_02A 65.0 133.6 130.3 L328_13S 64.5 130.6 123.2 P271_01G68.0 110.2 107.0 Q295_05P 64.3 106.3 103.3 Q295_07I 66.5 108.0 102.3S324_06M 66.1 123.1 117.1 S324_09D 66.2 112.8 105.7 S324_10E 66.9 104.1102.8 S324_20W 58.2 108.3 103.1 S337_14T 67.3 104.7 103.3 S337_16H 66.6101.5 101.4

TABLE 33-2 NAME Tm Ho/Con_2aH He/Con_2aH T335_02A 65.8 103.0 100.8T335_04F 64.6 103.7 103.1 T335_05P 57.1 115.0 107.6 T335_06M 66.2 106.5102.7 T335_08L 64.9 105.9 105.6 T335_09D 61.2 109.5 106.9 Y300_06M 61.9106.3 104.8 Y300_07I 60.5 122.3 108.4 Y300_08L 60.6 115.2 108.2

TABLE 34-1 NAME Tm Ho/Con_2b He/Con_2b A330_01G 67.0 102.1 103.5G237_04F 66.8 139.8 149.2 G237_20W 67.6 165.7 193.4 I332_14T 63.1 105.8106.4 I336_07I 67.9 100.6 103.4 I336_08L 64.8 103.0 107.4 K326_07I 66.1169.6 198.8 K326_08L 67.1 168.5 176.6 K326_20W 65.5 131.8 148.5 L235_20W67.9 116.3 147.3 L328_02A 65.0 116.7 144.5 L328_13S 64.5 146.6 149.2P238_04F 66.4 209.1 312.0 P238_09D 60.8 206.6 220.9 P238_15Y 65.5 114.2217.8 P331_15Y 63.2 110.7 114.4 P331_16H 63.0 100.4 116.0 P331_16H 61.8110.9 111.2 S239_03V 67.7 108.0 108.4 S239_10E 66.0 175.1 180.6 S267_06M66.4 186.7 209.5 S267_19Q 66.8 184.9 217.5 S324_06M 66.1 103.6 112.1S337_16H 66.6 102.7 104.6 T335_07I 67.0 104.3 106.1 T335_10E 62.9 107.7108.4 T335_14T 68.0 101.9 106.6 T335_15Y 64.7 103.9 108.2 V266_06M 64.8231.3 251.2 Y296_20W 66.7 102.6 103.3 A327_09D 65.7 174.7 171.7 A327_10E65.4 147.8 140.5 G236_09D 66.2 206.8 167.0 G236_10E 65.9 114.3 110.2H268_01G 67.5 157.6 143.6 H268_09D 67.1 297.5 239.1 H268_10E 67.6 274.9230.5 H268_13S 67.8 186.0 159.6 H268_18N 66.8 178.7 156.3 I332_04F 61.9117.1 104.0 I332_06M 65.0 106.4 104.7 I332_09D 56.2 181.7 147.0 I332_10E60.1 180.7 142.4 I332_15Y 59.4 108.9 101.3 K326_03V 66.5 183.5 175.3K326_04F 65.3 159.6 155.1 K326_06M 66.6 193.1 164.9 K326_09D 68.0 239.8192.9 K326_10E 68.0 237.2 200.1 K326_14T 64.1 177.0 149.5 K326_15Y 65.7162.5 159.4 K326_16H 66.7 131.2 118.7 K326_19Q 67.9 163.0 139.3 K334_02A65.8 109.5 107.5 K334_03V 67.0 145.2 124.7 K334_04F 65.9 115.0 110.9K334_05P 62.0 112.0 105.1 K334_07I 67.3 137.2 119.1 K334_08L 66.2 114.0106.5 K334_10E 63.6 129.1 114.5 K334_13S 66.1 117.3 110.9 K334_14T 65.7138.4 115.4

TABLE 34-2 NAME Tm Ho/Con_2b He/Con_2b K334_15Y 65.9 126.0 109.2K334_16H 62.8 123.8 112.8 K334_18N 65.4 128.0 114.8 K334_19Q 67.1 116.8109.3 L328_03V 64.9 135.3 125.7 L328_04F 67.2 241.1 218.8 L328_06M 67.7170.8 141.6 L328_07I 66.0 188.4 160.3 L328_14T 64.9 146.2 141.3 L328_15Y65.5 272.1 192.9 L328_19Q 62.8 110.7 104.9 P238_08L 67.4 235.3 204.5P271_01G 68.0 228.7 193.3 P271_08L 66.4 113.1 102.7 P331_04F 63.0 113.1110.5 S239_01G 64.9 173.5 144.2 S239_07I 67.3 108.1 106.8 S239_08L 67.6142.1 140.3 S239_09D 65.0 223.7 205.8 S239_18N 67.5 124.9 119.1 S267_02A66.7 274.1 243.3 S267_03V 66.0 206.0 192.3 S267_07I 65.7 213.4 192.4S267_08L 67.3 108.9 108.8 S267_09D 65.4 326.7 310.5 S267_10E 64.1 396.2379.3 S267_14T 67.8 105.5 104.4 S298_08L 65.5 114.7 108.5 S337_09D 56.5116.3 113.7 S337_10E 62.1 109.0 107.3 S337_14T 67.3 109.8 104.6 T335_04F64.6 106.8 102.9 T335_08L 64.9 106.0 103.9 T335_09D 61.2 116.0 108.4Y300_19Q 63.0 124.0 108.3

TABLE 35-1 NAME Tm Ho/Con_3a He/Con_3a I332_01G 56.8 103.6 110.7I336_02A 61.8 103.6 111.0 I336_03V 67.2 110.0 112.2 I336_06M 61.7 107.0108.9 I336_07I 67.9 100.2 103.1 I336_18N 62.0 100.6 104.0 K326_07I 66.1133.5 138.6 K326_08L 67.1 113.9 114.2 K334_01G 64.2 103.6 121.4 K334_09D65.6 149.7 159.7 Q295_14T 65.9 108.2 114.7 S267_02A 66.7 145.3 150.4S267_09D 65.4 159.7 180.8 S298_14T 66.1 106.7 112.6 S337_09D 56.5 105.5109.9 S337_10E 62.1 100.2 105.0 S337_18N 64.1 101.5 104.1 T335_07I 67.0107.0 107.2 T335_10E 62.9 102.4 106.9 T335_14T 68.0 102.5 108.5 A330_04F67.3 174.3 147.1 A330_08L 66.9 138.4 124.5 A330_20W 67.9 117.9 104.2E333_16H 60.8 110.9 103.0 H268_09D 67.1 220.3 197.2 H268_10E 67.6 205.5186.1 H268_13S 67.8 129.6 121.9 I332_02A 63.2 114.1 112.3 I332_09D 56.2254.6 206.6 I332_10E 60.1 278.5 232.7 I332_13S 60.9 112.3 104.5 I332_14T63.1 138.3 119.8 I332_15Y 59.4 105.6 101.0 I332_19Q 62.8 118.4 110.5I336_09D 53.5 110.9 106.3 I336_10E 53.8 130.0 119.4 I336_13S 57.4 119.4101.6 I336_14T 64.1 122.7 108.3 K326_03V 66.5 137.0 122.1 K326_04F 65.3102.9 102.9 K326_06M 66.6 128.9 114.8 K326_09D 68.0 162.7 133.6 K326_10E68.0 157.2 124.5 K326_14T 64.1 147.4 126.5 K326_15Y 65.7 115.4 112.3K326_19Q 67.9 117.7 106.3 K334_02A 65.8 154.2 147.2 K334_03V 67.0 169.7149.5 K334_04F 65.9 168.0 147.5 K334_05P 62.0 164.6 133.7 K334_07I 67.3179.9 151.1 K334_08L 66.2 178.0 143.3 K334_10E 63.6 206.0 180.6 K334_13S66.1 153.2 145.3 K334_14T 65.7 163.4 141.4 K334_15Y 65.9 180.9 140.8K334_16H 62.8 152.9 133.7 K334_18N 65.4 155.9 136.2 K334_19Q 67.1 149.5136.1 Q295_02A 66.5 107.5 105.6 Q295_10E 64.9 111.2 100.2 S239_09D 65.0192.1 165.5

TABLE 35-2 NAME Tm Ho/Con_3a He/Con_3a S239_10E 66.0 251.9 181.4S239_18N 67.5 115.9 109.9 S298_03V 65.0 110.1 109.3 S324_01G 65.4 119.4104.9 S324_06M 66.1 114.1 112.7 S324_09D 66.2 101.1 101.0 S324_14T 67.1108.5 103.1 S324_20W 58.2 101.9 101.8 S337_01G 64.9 108.1 102.2 S337_16H66.6 112.3 107.7 T335_05P 57.1 118.8 104.9 T335_06M 66.2 107.2 101.5T335_08L 64.9 102.9 102.4 T335_09D 61.2 113.8 107.7 Y296_20W 66.7 145.7135.0 Y300_08L 60.6 104.9 104.9

[Example 11] ADCC Activity Assay of Heterodimerized Antibodies withImproved Ability to Recognize FcγRIIIa

As discussed in Example 7, by using the heterodimerized antibody, theFcγRIIIa-binding activity was successfully increased more than that ofvariants produced by the conventional homodimerized antibody technology.Antibodies induce NK cells via FcγRIIIa to exhibit antibody-dependentcellular cytotoxicity against cells expressing the target antigen. Toassess whether the heterodimerized antibodies have not only increasedFcγRIIIa-binding activity but also increased ADCC activity, theheterodimerized antibodies with increased FcγRIIIa-binding activityshown in Table 13 of Example 7, homodimerized antibodies, and nativeIgG1 were assayed for ADCC activity according to the method described inReference Example 7. The result is shown in FIG. 31.

Based on the result shown in FIG. 31, when GpH7-G1d/GpL16-k0 which is anative IgG1 is compared to GpH7-A5/GpH7-B3/GpL16-k0 in which D356K,H435R, and K439E each was introduced into one H chain, there is nosignificant difference in the ADCC activity. Thus, it was thought thatthe alterations D356K, H435R, and K439E do not affect the ADCC activity.

Then, homodimerized antibodies in which an alteration that increases theFcγRIIIa-binding activity was introduced into both H chains in theconventional way, were examined to assess whether the same tendency asseen in the binding enhancing effect is also observed for the ADCCactivity. GpH7-TA7/GpH7-TA45/GpL16-k0 in which both of the H chains wereintroduced with L234Y, G236W, and S298A was compared toGpH7-A57/GpH7-B78/GpL16-k0 in which both of the H chains were introducedwith S239D, A330L, and I332E. Regarding the FcγRIIIa-binding activity,the binding of GpH7-A57/GpH7-B78/GpL16-k0 was markedly enhanced ascompared to GpH7-A5/GpH7-B3/GpL16-k0, while the binding ofGpH7-TA7/GpH7-TA45/GpL16-k0 was reduced. Likewise, the ADCC activity ofGpH7-A57/GpH7-B78/GpL16-k0 was increased more than that ofGpH7-A5/GpH7-B3/GpL16-k0, while the activity ofGpH7-TA7/GpH7-TA45/GpL16-k0 was reduced compared toGpH7-A5/GpH7-B3/GpL16-k0. Thus, as to the homodimerized antibodies, acorrelation was observed between the level of the FcγRIIIa-bindingactivity and that of the ADCC activity.

Then, heterodimerized antibodies in which only one of the H chains wasintroduced with an alteration that increases the FcγRIIIa-bindingactivity were examined to assess whether the same tendency as seen inthe binding enhancing effect is observed for the ADCC activity.GpH7-TA7/GpH7-B3/GpL16-k0 in which one of the H chains was introducedwith L234Y, G236W, and S298A was compared to GpH7-A5/GpH7-B78/GpL16-k0in which one of the H chains was introduced with S239D, A330L, andI332E. The FcγRIIIa-binding activities of GpH7-A5/GpH7-B78/GpL16-k0 andGpH7-TA7/GpH7-B3/GpL16-k0 were increased as compared toGpH7-A5/GpH7-B3/GpL16-k0. The same tendency was observed for the ADCCactivity. Furthermore, the FcγRIIIa-binding activity ofGpH7-A5/GpH7-B78/GpL16-k0 was increased more than that ofGpH7-TA7/GpH7-B3/GpL16-k0; however, the same tendency was obtained forADCC activity. Thus, not only the homodimerized antibodies but also theheterodimerized antibodies have a correlation between the level of theFcγRIIIa-binding activity and the ADCC activity.

Then, regarding each of the groups of the alterations L234Y, G236W, andS298A, and the alterations S239D, A330L, and I332E, it was assessedwhether heterodimerized antibodies and homodimerized antibodies have acorrelation between the effects to enhance the FcγRIIIa-binding activityand ADCC activity. First, when GpH7-A5/GpH7-B78/GpL16-k0 which is aheterodimerized antibody in which only one the H chains was introducedwith the group of the alteration S239D, A330L, and I332E was compared toGpH7-A57/GpH7-B78/GpL16-k0 which is a homodimerized antibody in whichboth of the H chains were introduced with the group, the effect toenhance the FcγRIIIa-binding activity was greater in the homodimerizedantibody than the heterodimerized antibody; however, there was nodifference for the ADCC activity. Furthermore, whenGpH7-TA7/GpH7-B3/GpL16-k0 which is a heterodimerized antibody in whichonly one of the H chains was introduced with the group of thealterations L234Y, G236W, and S298A was compared toGpH7-TA7/GpH7-TA45/GpL16-k0 which is a homodimerized antibody in whichboth of the H chains were introduced with the group, with respect to theFcγRIIIa-binding activity, the binding of the heterodimerized antibodywas enhanced more strongly than that of GpH7-A5/GpH7-B3/GpL16-k0,whereas the binding of the homodimerized antibody was reduced. The sametendency was observed for the ADCC activity. Thus, it was thought thatthe effect of the group of the alterations L234Y, G236W, and S298A toincrease the FcγRIIIa-binding activity from only one direction isreflected in the ADCC activity. From these results, it was thought thatin heterodimerized antibodies in which only one of the H chains has beenintroduced with a group of alterations, and homodimerized antibodies inwhich both of the H chains have been introduced with the group, there isa correlation between the level of the FcγRIIIa-binding activity and theADCC activity.

Next, the heterodimerized antibody GpH7-TA7/GpH7-B78/GpL16-k0 in whichone of the H chains was introduced with L234Y, G236W, and S298A and theother H chain was introduced with S239D, A330L, and I332E was comparedto the homodimerized antibody GpH7-A57/GpH7-B78/GpL16-k0 in which bothof the H chains were introduced with S239D, A330L, and I332E. Regardingthe FcγRIIIa-binding activity, both had markedly increased bindingactivities as compared to GpH7-A5/GpH7-B3/GpL16-k0. The same tendencywas observed for the ADCC activity. In addition, the FcγRIIIa-bindingactivity of GpH7-TA7/GpH7-B78/GpL16-k0 was increased more than that ofGpH7-A57/GpH7-B78/GpL16-k0, and GpH7-TA7/GpH7-B78/GpL16-k0 alsoexhibited a stronger ADCC activity.

As described above, regarding the groups of the alterations L234Y,G236W, and S298A, and the alterations S239D, A330L, and I332E, thelatter alteration group, S239D, A330L, and I332E, was observed to have astronger effect to increase the ADCC activity regardless of whenintroduced into one H chain or both H chains. On the other hand, it wasdemonstrated that, when the group of alterations L234Y, G236W, andS298A, and the group of the alterations S239D, A330L, and I332E arerespectively introduced into different H chains, the effect to increasethe ADCC activity is stronger than when S239D, A330L, and I332E, whichhave a strong effect to increase the ADCC activity in bothheterodimerized and homodimerized antibodies, are introduced into both Hchains.

That is, it was demonstrated that the correlation between the level ofthe FcγRIIIa-binding activity and the ADCC level, such as that observedin homodimerized antibodies of the prior art technology, is alsoobserved when comparing heterodimerized antibodies to one another andwhen comparing heterodimerized and homodimerized antibodies. Thisreveals that the use of the heterodimerized antibody technology enablesthe preparation of antibodies having ADCC activity superior to those byconventional technologies.

[Example 12] Comparison of Conventional Homodimerized Antibodies andNovel Heterodimerized Antibodies in Connection with FcγRIIa

As described in Example 1, FcγRIIIa is thought to play an important rolein the drug efficacy of antibody pharmaceuticals. Furthermore, attentionhas been drawn on the role of FcγRIIa that is played in the drugefficacy of IgG1-derived antibody pharmaceuticals, in addition toFcγRIIIa.

For FcγRIIa, there are allotypes called R and H types, which have Argand His at amino acid position 131, respectively, and they are known todiffer in the human IgG2-binding activity (Tissue Antigens 2003, 61,189-202). The susceptibility to infection is known to vary depending onthe difference in the FcγRIIa allotype (Tissue Antigens2003:61:189-202). This is thought to be because the IgG2-bindingactivity of FcγRIIa varies depending on the difference in the allotype,and as a result the mechanism of resistance to pathogens via IgG2differs (Infection and Immunity 1995, 63, 73-81). Meanwhile, cellsexpressing mouse FcγRIV are known to correspond to cells expressinghuman FcγRIIa, and FcγRIV has been reported to play an important role inthe drug efficacy of an anti-CD20 antibody in mouse model. Thesefindings suggest that, in human, FcγRIIa plays a similar role (TheJournal of Experimental Medicine 2004:199:1659-1669; The Journal ofExperimental Medicine 2006, 203, 743-753; Immunity 2005, 23, 41-51).Indeed, it has been reported that, an antibody in which theFcγRIIa-binding activity of the Fc region is enhanced compared with IgG1enhances the macrophage-mediated antibody-dependent cellularphagocytosis (ADCP) activity as compared to IgG1 (Molecular CancerTherapeutics 2008, 7, 2517-2527). Furthermore, in a mouse xenograftmodel, an anti-CD19 antibody having an Fc domain with enhanced ADCPexhibits an antitumor effect stronger than that of IgG1 (Nature Medicine2000, 6, 443-446). The Fc domain of this antibody has increased bindingactivity to monkey FcγRIIa. CD19 is expressed on B cell surface. It hasbeen reported that this antibody, when administered to monkeys, enhancesthe B cell elimination as compared to an anti-CD19 antibody having theFc region of IgG1 (Science 2005, 310, 1510-1512).

From these reports, it is expected that the efficacy of antibodypharmaceuticals, in particular, the antitumor effect can be furtherimproved by increasing the FcγRIIa-binding activity in addition toenhancing the FcγRIIIa-binding activity. Antibodies with suchcharacteristics have been produced using conventional technologies(Molecular Cancer Therapeutics 2008, 7, 2517-2527). However, sinceFcγRIIa is thought to bind to an antibody Fc domain in an asymmetricmanner, it is considered that the FcγRIIa-binding activity can befurther increased by using the heterodimerized antibody technology asdescribed in Example 11. To assess this, alterations that increase thebinding activity to all of FcγRIIIa, FcgRIIa R type, and FcgRIIa H typeas compared to native IgG were selected based on the result shown inExample 4, and combined them to introduce the mutations that enhancingthe binding activity to all of FcγRIIIa, FcgRIIa R type, and FcgRIIa Htype, and heterodimerized antibodies were produced to have a combinationof H chains with differential FcγR binding. The antibodies were assessedfor the binding activity to each FcγR.

Meanwhile, in contrast to these activating FcγR, FcγRIIb which is aninhibitory FcγR induces intracellular signal that suppresses the immuneresponse. It has been reported that in FcγRIIb-knockout mice theantitumor effect of antibodies is enhanced (Nature Medicine 2000, 6,443-446) and antibody-mediated B cell elimination is promoted (TheJournal of Experimental Medicine 2006, 203, 743-753), showing thatFcγRIIb plays an important role in the drug efficacy of antibodies invivo. Meanwhile, a correlation has been observed between the antitumoreffect of mouse IgG subclass and the ratio of the binding activity toactivating FcγR against the binding activity to inhibitory FcγR (A/Iratio) of each IgG subclass (Science 2005, 310, 1510-1512). Thesereports suggest that A/I ratios are important for antibody effectorfunction via immunity. Specifically, when antibodies are produced tohave higher A/I ratio, their effector function is enhanced, and suchantibodies are useful. It was, however, predicted to be extremelydifficult to increase the A/I ratio by increasing the FcγRIIa-bindingactivity without increasing the FcγIIb-binding activity, because thesequence homology between FcγRIIa which is an activating FcγR, andFcγRIIb which is an inhibitory FcγR is 93%, which is extremely high, attheir extracellular domains. Like FcγRIIIa and FcγRIIa, FcγRIIb isthought to bind to an antibody Fc domain in an asymmetric manner. Withconventional technologies, the interaction with FcγR can be regulatedonly by introducing the same alteration into both H chains of anantibody. In contrast, the use of the heterodimerized antibodytechnology enables more precise regulation, and thus enables improvementof the A/I ratio even between FcγRIIa and FcγRIIb whose sequences arehighly similar. Thus, the present inventors also assessed, from thisviewpoint, whether the heterodimerized antibody technology is superiorto conventional technologies.

In this assessment, to efficiently form heterodimerized antibodies, theknobs-into-holes technology was used for the antibody H chain constantregion. The knobs-into-holes technology is a technology that enablespromotion of heterodimerization of H chains and efficient preparation ofheterodimerized antibodies of interest by substituting an amino acidside chain with a larger side chain (knob) in the CH3 domain of one Hchain and substituting an amino acid side chain with a smaller sidechain (hole) in the CH3 domain of the other H chain so that the knob isplaced in the hole (Nature, 372, 379-383 (1994)). An H chain whoseconstant region has been introduced with the alterations Y349C and T366Wthat intend to have larger amino acid side chains in the CH3 domain isreferred to as “knob chain”. When an additional alteration is introducedinto this, the name of the H chain constant region begins with symbol“Kn”, which is followed by three-digit number, such as Kn001. An H chainwhose constant region has been introduced with the alterations D356C,T366S, L368A, and Y407V that intend to have smaller amino acid sidechains in the CH3 domain is referred to as “hole chain”. When anadditional alteration is introduced into this, the name of the H chainconstant region begins with symbol “H1”, which is followed bythree-digit number, and thus is referred to as, such as H1001.Furthermore, when the antibody H chain constant regions are Kn001 andH1001, the sequences of the H chains of an antibody whose variableregions have GpH7 are referred to as GpH7-Kn001 and GpH7-H1001,respectively. An antibody purified after expression is referred to, forexample, as GpH7-Kn001/GpH7-H1001/GpL16-k0 when the sequencecorresponding to an antibody H chain used to express the heterodimerizedantibody is GpH7-Kn001, and the sequence corresponding to the otherantibody H chain is GpH7-H1001, and the sequence corresponding to theantibody L chain is GpL16-k0.

First, GpH7-Kn033 (SEQ ID NO: 51) resulting from the introduction of thealterations Y349C and T366W into the constant region for GpH7-G1d, andGpH7-H1033 (SEQ ID NO: 56) resulting from the introduction of thealterations D356C, T366S, L368A, and Y407V into constant region forGpH7-G1d were constructed according to the method described in ReferenceExample 1. When expressing heterodimerized antibodies, the followingexpression vectors were used for efficient expression of them: anexpression vector inserted with GpL16-k0 for the antibody L chain; forone antibody H chain, an expression vector inserted with a sequenceintroduced with a further alteration to GpH7-Kn033 (SEQ ID NO: 51)introduced with the alterations Y349C and T366W; and, for the otherantibody H chain, an expression vector inserted with a sequenceintroduced with a further alteration to GpH7-H1033 (SEQ ID NO: 56)introduced with the alterations D356C, T366S, L368A, and Y407V.

Antibodies whose binding to all of FcγRIIIa, FcγRIIa R type, and FcγRIIaH type is intended to be enhanced were produced as described below,based on the information obtained in Example 4 on the influence of eachalteration on the binding of an antibody to each FcγR. When introducingdifferent alterations into the constant regions of respective H chainsof an antibody, GpH7-Kn033 and GpH7-H1033 were used as parentalpolypeptides. GpH7-Kn045 (SEQ ID NO: 54) resulting from the introductionof L234Y, L235Y, G236A, H268D, and S298A into GpH7-Kn033; GpH7-Kn056(SEQ ID NO: 55) resulting from the introduction of L234Y, L235Y, G236A,H268D, Q295L, and S298A into GpH7-Kn033; GpH7-H1048 (SEQ ID NO: 59)resulting from the introduction of G236A, S239D, A330K, and I332E intoGpH7-H1033; and GpH7-H1055 (SEQ ID NO: 60) resulting from theintroduction of G236A, S239D, Q295L, A330M, and I332E into GpH7-H1033,were constructed according to the method described in Reference Example1.

Then, respective H chains were combined in the manner described below,and the antibodies were expressed according to Reference Example 1.GpH7-Kn033/GpH7-H1033/GpL16-k0 resulting from the application of theknobs-into-holes technology alone to G1d was expressed using GpH7-Kn033and GpH7-H1033 as the H chain and GpL16-k0 as the L chain. Theheterodimerized antibody GpH7-Kn045/GpH7-H1048/GpL16-k0 was expressedusing GpH7-Kn045 and GpH7-H1048 as the H chain and GpL16-k0 as the Lchain. The heterodimerized antibody GpH7-Kn045/GpH7-H1055/GpL16-k0 wasexpressed using GpH7-Kn045 and GpH7-H1055 as the H chain and GpL16-k0 asthe L chain. The heterodimerized antibody GpH7-Kn056/GpH7-H1055/GpL16-k0was expressed using GpH7-Kn056 and GpH7-H1055 as the H chain andGpL16-k0 as the L chain.

Antibodies based on the use of conventional technologies for comparisonwere prepared with reference to Pro. Nat. Acad. Sci., 103, 4005-4010(2006) as described below. The alteration G236A/S239D/I332E, which hasbeen reported to enhance the binding to all of FcγRIIIa, FcγRIIa R type,and FcγRIIa H type, was introduced into GpH7-Kn033 and GpH7-H1033 toconstruct GpH7-Kn037 (SEQ ID NO: 52) and GpH7-H1036 (SEQ ID NO: 57),respectively. Furthermore, the alteration S239D/A330L/I332E, which hasbeen reported to enhance the FcγRIIIa binding, was introduced intoGpH7-Kn033 and GpH7-H1033 to construct GpH7-Kn032 (SEQ ID NO: 53) andGpH7-H1032 (SEQ ID NO: 58), respectively. These H chains were combined,and antibodies were expressed according to Reference Example 1. Thehomodimerized antibody GpH7-Kn037/GpH7-H1036/GpL16-k0, resulting fromthe introduction of G236A/S239D/I332E into both H chains ofGpH7-Kn033/GpH7-H1033/GpL16-k0, which is a molecule resulting from theapplication of the knobs-into-holes technology alone to G1d, wasexpressed using GpH7-Kn037 and GpH7-H1036 as the H chain and GpL16-k0 asthe L chain. The homodimerized antibody GpH7-Kn032/GpH7-H1032/GpL16-k0,resulting from the introduction of S239D/A330L/I332E into both H chainsof GpH7-Kn033/GpH7-H1033/GpL16-k0, which is a molecule resulting fromthe application of the knobs-into-holes technology alone to G1d, wasexpressed using GpH7-Kn032 and GpH7-H1032 as the H chain and GpL16-k0 asthe L chain.

These antibodies were assessed for the binding activity to each FcγRaccording to the method described in Reference Example 2. The result issummarized in Table 36. Meanwhile, the KD ratio of each antibody isshown in Table 37, and the A/I ratio, which is the KD ratio forFcγRIIIa, is summarized in Table 38.

TABLE 36 MUTATION MUTATION FcgRIa FcgRIIa H FcgRIIa R FcgRIIb FcgRIIIa FFcgRIIIa V SAMPLE Knob SITE Hole SITE KD (M) KD (M) KD (M) KD (M) KD (M)KD (M) GpH7-Kn033/GpH7- Kn033 — Hl033 — 6.9E−11 5.9E−07 7.4E−07 3.6E−061.6E−06 2.2E−07 Hl033/GpL16-k0 (SEQ ID NO: 51, 56, 5) GpH7-Kn037/GpH7-Kn037 G236A/ Hl036 G236A/ 1.2E−11 2.7E−08 1.7E−08 2.3E−07 9.7E−092.6E−09 Hl036/GpL16-k0 S239D/ S239D/ (SEQ ID NO: I332E I332E 52, 57, 5)GpH7-Kn032/GpH7- Kn032 S239D/ Hl032 S239D/ 2.8E−12 4.7E−07 2.5E−074.4E−07 4.1E−09 1.2E−09 Hl032/GpL16-k0 A330L/ A330L/ (SEQ ID NO: I332EI332E 53, 58, 5) GpH7-Kn045/GpH7- Kn045 L234Y/ Hl048 G236A/ 1.9E−111.1E−08 4.8E−09 1.1E−07 3.7E−09 1.3E−09 Hl048/GpL16-k0 L235Y/ S239D/(SEQ ID NO: G236A/ A330K/ 54, 59, 5) H268D/ I332E S298A GpH7-Kn045/GpH7-Kn045 L234Y/ Hl055 G236A/ 1.9E−11 2.9E−08 1.3E−08 2.4E−07 1.6E−097.2E−10 Hl055/GpL16-k0 L235Y/ S239D/ (SEQ ID NO: G236A/ Q295L/ 54, 60,5) H268D/ A330M/ S298A I332E GpH7-Kn056/GpH7- Kn056 L234Y/ Hl055 G236A/2.3E−11 3.0E−08 1.7E−08 2.6E−07 1.4E−09 6.6E−10 Hl055/GpL16-k0 L235Y/S239D/ (SEQ ID NO: G236A/ Q295L/ 55, 60, 5) H268D/ A330M/ Q295L/ I332ES298A

The table shows the binding activity to each FcγR of heterodimerizedantibodies with enhanced binding to FcγRIIa and Ma.

The column “SAMPLE” indicates antibody names; the columns “Kn” and “H1”indicate names of the constant regions of the knob chain and hole chainof each antibody; and the column “MUTATION SITE” indicates mutationsthat are different in comparison with GpH7-Kn033/GpH7-H1033/GpL16-k0(“-”: when there is no particular mutation).

TABLE 37 FcgRIa FcgRIIa H FcgRIIa R FcgRIIb FcgRIIIa F FcgRIIIa VMUTATION MUTATION KD KD KD KD KD KD SAMPLE Knob SITE Hole SITE ratioratio ratio ratio ratio ratio GpH7-Kn033/GpH7- Kn033 — Hl033 — 1 1 1 1 11 Hl033/GpL16-k0 (SEQ ID NO: 51, 56, 5) GpH7-Kn037/GpH7- Kn037 G236A/Hl036 G236A/ 6 22 43 16 161 84 Hl036/GpL16-k0 S239D/ S239D/ (SEQ ID NO:I332E I332E 52, 57, 5) GpH7-Kn032/GpH7- Kn032 S239D/ Hl032 S239D/ 24 1.23.0 8.2 381 182 Hl032/GpL16-k0 A330L/ A330L/ (SEQ ID NO: I332E I332E 53,58, 5) GpH7-Kn045/GpH7- Kn045 L234Y/ Hl048 G236A/ 3.7 52 154 34 419 170Hl048/GpL16-k0 L235Y/ S239D/ (SEQ ID NO: G236A/ A330K/ 54, 59, 5) H268D/I332E S298A GpH7-Kn045/GpH7- Kn045 L234Y/ Hl055 G236A/ 3.7 21 56 15 985300 Hl055/GpL16-k0 L235Y/ S239D/ (SEQ ID NO: G236A/ Q295L/ 54, 60, 5)H268D/ A330M/ S298A I332E GpH7-Kn056/GpH7- Kn056 L234Y/ Hl055 G236A/ 2.920 44 14 1114 327 Hl055/GpL16-k0 L235Y/ S239D/ (SEQ ID NO: G236A/ Q295L/55, 60, 5) H268D/ A330M/ Q295L/ I332E S298A

The table shows the binding activity to each FcγR of heterodimerizedantibodies with enhanced binding to FcγRIIa and Ma.

The column “SAMPLE” indicates antibody names; the columns “Kn” and “H1”indicate names of the constant regions of the knob chain and hole chainof each antibody; and the column “MUTATION SITE” indicates mutationsthat are different in comparison with GpH7-Kn033/GpH7-H1033/GpL16-k0(“-”: when there is no particular mutation). A value obtained bydividing KD of GpH7-Kn033/GpH7-H1033/GpL16-k0 for FcγR by the KD of eachantibody was defined as “KD ratio”. The SEQ ID NOs are also shown forthe amino acid sequences of the H chain and L chain of each antibody.

TABLE 38 MUTATION MUTATION A/I ratio SAMPLE Knob SITE Hole SITE FcgRIIaH FcgRIIa R GpH7-Kn033/GpH7- Kn033 — Hl033 — 6.2 4.9 Hl033/GpL16-k0 (SEQID NO: 51, 56, 5) GpH7-Kn037/GpH7- Kn037 G236A/S239D/ Hl036 G236A/S239D/8.6 13 Hl036/GpL16-k0 I332E I332E (SEQ ID NO: 52, 57, 5)GpH7-Kn032/GpH7- Kn032 S239D/A330L/ Hl032 S239D/A330L/ 0.93 1.8Hl032/GpL16-k0 I332E I332E (SEQ ID NO: 53, 58, 5) GpH7-Kn045/GpH7- Kn045L234Y/L235Y/ Hl048 G236A/S239D/ 9.5 22 Hl048/GpL16-k0 G236A/H268D/A330K/I332E (SEQ ID NO: 54, 59, 5) S298A GpH7-Kn045/GpH7- Kn045L234Y/L235Y/ Hl055 G236A/S239D/ 8.3 18 Hl055/GpL16-k0 G236A/H268D/Q295L/A330M/ (SEQ ID NO: 54, 60, 5) S298A I332E GpH7-Kn056/GpH7- Kn056L234Y/L235Y/ Hl055 G236A/S239D/ 8.7 16 Hl055/GpL16-k0 G236A/H268D/Q295L/A330M/ (SEQ ID NO: 55, 60, 5) Q295L/S298A I332E

The table shows the ratio between the binding activity to activatingFcγR and the binding activity to inhibitory FcγR.

The column “SAMPLE” indicates antibody names; the columns “Kn” and “H1”indicate names of the constant regions of the knob chain and hole chainof each antibody; and the column “MUTATION SITE” indicates mutationsthat are different in comparison with GpH7-Kn033/GpH7-H1033/GpL16-k0(“-”: when there is no particular mutation). A value obtained bydividing KD of GpH7-Kn033/GpH7-H1033/GpL16-k0 for FcγRIIb by the KD ofeach antibody for FcγRIIa H type and R type was defined as “A/I ratio”for each type. The SEQ ID NOs are also shown for the amino acidsequences of the H chain and L chain of each antibody.

The results shown in Tables 36 and 37 demonstrate that, regardingGpH7-Kn037/GpH7-H1036/GpL16-k0 in which both of the H chains wereintroduced with G236A/S239D/I332E, its binding to FcγRIIa type H wasenhanced by 22 times, its binding to FcγRIIa type R was enhanced by 43times, and its binding to FcγRIIIa F was enhanced by 161 times ascompared to GpH7-Kn033/GpH7-H1033/GpL16-k0 which is a molecule resultingfrom the application of the knobs-into-holes technology alone to G1d.Meanwhile, the result shown in Table 38 demonstrates that the A/I ratioof GpH7-Kn037/GpH7-H1036/GpL16-k0 was 8.6 for FcγRIIa type H and 13 forFcγRIIa type R, which were increased as compared to, respectively, 6.2and 4.9 of GpH7-Kn033/GpH7-H1033/GpL16-k0.

The results shown in Tables 36 and 37 demonstrate that, regardingGpH7-Kn032/GpH7-H1032/GpL16-k0 in which both of the H chains wereintroduced with S239D/A330L/I332E, its binding to FcγRIIa type H wasenhanced by 1.2 times, its binding to FcγRIIa type R was enhanced by 3.0times, and its binding to FcγRIIIa F was enhanced by 381 times ascompared to GpH7-Kn033/GpH7-H1033/GpL16-k0 which is a molecule resultingfrom the application of the knobs-into-holes technology alone to G1d.Meanwhile, the result shown in Table 38 demonstrates that the A/I ratioof GpH7-Kn032/GpH7-H1032/GpL16-k0 was 0.93 for FcγRIIa type H and 1.8for FcγRIIa type R, which were decreased as compared toGpH7-Kn033/GpH7-H1033/GpL16-k0.

The results shown in Tables 36 and 37 demonstrate that, regardingGpH7-Kn045/GpH7-H1048/GpL16-k0 in which one of the H chains wasintroduced with L234Y/L235Y/G236A/H268D/S298A and the other H chain withG236A/S239D/A330K/I332E, its binding to FcγRIIa type H was enhanced by52 times, its binding to FcγRIIa type R was enhanced by 154 times, andits binding to FcγRIIIa F was enhanced by 419 times as compared toGpH7-Kn033/GpH7-H1033/GpL16-k0 which is a molecule resulting from theapplication of the knobs-into-holes technology alone to G1d. The resultalso shows that, for both H and R types, the FcγRIIa-binding activitywas increased as compared to the homodimerized antibodyGpH7-Kn037/GpH7-H1036/GpL16-k0 in which both of the H chains wereintroduced with G236A/S239D/I332E of the prior art technology. Inaddition, the FcγRIIIa F-binding activity was slightly increased ascompared to the homodimerized antibody GpH7-Kn032/GpH7-H1032/GpL16-k0 inwhich both of the H chains were introduced with S239D/A330L/I332E of theprior art technology. The result shown in Table 38 demonstrates that theA/I ratio of GpH7-Kn045/GpH7-H1048/GpL16-k0 was 9.5 for FcγRIIa type Hand 22 for FcγRIIa type R, which were increased when compared to any ofGpH7-Kn033/GpH7-H1033/GpL16-k0, GpH7-Kn037/GpH7-H1036/GpL16-k0, andGpH7-Kn032/GpH7-H1032/GpL16-k0. The result demonstrates that, using theheterodimerized antibody technology, the FcγRIIa- and FcγRIIIa F-bindingactivities of GpH7-Kn045/GpH7-H1048/GpL16-k0 are increased and theantibody binds more selectively to activating FcγR, as compared to whenusing the conventional technology.

The results shown in Tables 36 and 37 demonstrate that, regardingGpH7-Kn045/GpH7-H1055/GpL16-k0 in which one of the H chains wasintroduced with L234Y/L235Y/G236A/H268D/S298A and the other H chain wasintroduced with G236A/S239D/Q295L/A330M/I332E, its binding to FcγRIIatype H was enhanced by 21 times, its binding to FcγRIIa type R wasenhanced by 56 times, and its binding to FcγRIIIa F was enhanced by 985times as compared to GpH7-Kn033/GpH7-H1033/GpL16-k0 which is a moleculeresulting from the application of the knobs-into-holes technology aloneto G1d. The result also shows that, for both H and R types, theFcγRIIa-binding activity was almost equivalent to that of thehomodimerized antibody GpH7-Kn037/GpH7-H1036/GpL16-k0 in which both ofthe H chains were introduced with G236A/S239D/I332E of the prior arttechnology. The FcγRIIIa F-binding activity was increased as compared tothe homodimerized antibody GpH7-Kn032/GpH7-H1032/GpL16-k0 in which bothof the H chains were introduced with S239D/A330L/I332E of the prior arttechnology. The result shown in Table 38 demonstrates that the A/I ratioof GpH7-Kn045/GpH7-H1055/GpL16-k0 was 8.3 for FcγRIIa type H and 18 forFcγRIIa type R, which was increased as compared toGpH7-Kn033/GpH7-H1033/GpL16-k0 and GpH7-Kn032/GpH7-H1032/GpL16-k0; andas compared to GpH7-Kn037/GpH7-H1036/GpL16-k0, it was almost equivalentfor FcγRIIa type H and increased for FcγRIIa type R. This resultdemonstrates that, using the heterodimerized antibody technology, inGpH7-Kn045/GpH7-H1055/GpL16-k0, the FcγRIIIa-binding activity is furtherincreased while its FcγRIIa-binding activity is increased to acomparable extent, and the antibody binds more selectively to activatingFcγR, as compared to when using the conventional technology.

The results shown in Tables 36 and 37 demonstrate that, regardingGpH7-Kn056/GpH7-H1055/GpL16-k0 in which one of the H chains wasintroduced with L234Y/L235Y/G236A/H268D/Q295L/S298A and the other Hchain was introduced with G236A/S239D/Q295L/A330M/I332E, its binding toFcγRIIa type H was enhanced by 20 times, its binding to FcγRIIa type Rwas enhanced by 44 times, and its binding to FcγRIIIa F was enhanced by1114 times as compared to GpH7-Kn033/GpH7-H1033/GpL16-k0 which is amolecule resulting from the application of the knobs-into-holestechnology alone to G1d. This result also shows that, for both H and Rtypes, the FcγRIIa-binding activity was equivalent to that of thehomodimerized antibody GpH7-Kn037/GpH7-H1036/GpL16-k0 in which both ofthe H chains were introduced with G236A/S239D/I332E of the prior arttechnology. The FcγRIIIa F-binding activity was increased as compared tothe homodimerized antibody GpH7-Kn032/GpH7-H1032/GpL16-k0 in which bothof the H chains were introduced with S239D/A330L/I332E of the prior arttechnology. The result shown in Table 38 demonstrates that the A/I ratioof GpH7-Kn056/GpH7-H1055/GpL16-k0 was 8.7 for FcγRIIa type H and 16 forFcγRIIa type R, which was increased as compared toGpH7-Kn033/GpH7-H1033/GpL16-k0 and GpH7-Kn032/GpH7-H1032/GpL16-k0; andas compared to GpH7-Kn037/GpH7-H1036/GpL16-k0, it was almost equivalentfor FcγRIIa type H and increased for FcγRIIa type R. The resultdemonstrates that, using the heterodimerized antibody technology, inGpH7-Kn056/GpH7-H1055/GpL16-k0, the FcγRIIIa-binding activity is furtherincreased while its FcγRIIa-binding activity is increased to acomparable extent, and the antibody binds more selectively to activatingFcγR, as compared to when using the conventional technology.

[Example 13] Comparison with Conventional Technology: Assessment ofThermal Stability of Heterodimerized Antibodies with Increased FcγRIIa-and FcγRIIIa-Binding Activity

As described in Example 9, homodimerized antibodies obtained by theconventional technology, though having increased FcγR-binding activity,are physicochemically unstable, and this has reduced their value aspharmaceuticals. On the other hand, it was revealed that theheterodimerized antibody technology was convenient for regulating theeffect of each alteration to enhance the FcγR-binding activity and theinfluence from the physicochemical aspect, and that enhancement ofFcγR-binding activity without reducing the physicochemical stability waspossible. This Example tests whether antibodies with enhanced bindingactivities to FcγRIIa and FcγRIIIa, which are activating FcγR, similarlydo not have reduced physicochemical stability, thermodynamic stabilityin particular. Each antibody assessed for its FcγR-binding activity inExample 11 was assayed for the Tm of CH2 region according to the methoddescribed in Reference Example 5. The result is summarized in Table 39.

TABLE 39 MUTATION MUTATION Tm Sample Knob SITE Hole SITE (° C.)GpH7-Kn033/ Kn033 — Hl033 — 67.6 GpH7-Hl033/ GpL16-k0 (SEQ ID NO: 51,56, 5) GpH7-Kn037/ Kn037 G236A/ Hl036 G236A/ GpH7-Hl036/ S239D/ S239D/GpL16-k0 I332E I332E (SEQ ID NO: 52, 57, 5) GpH7-Kn032/ Kn032 S239D/Hl032 S239D/ 48.9 GpH7-Hl032/ A330L/ A330L/ GpL16-k0 I332E I332E (SEQ IDNO: 53, 58, 5) GpH7-Kn045/ Kn045 L234Y/ Hl048 G236A/ 60.9 GpH7-Hl048/L235Y/ S239D/ GpL16-k0 G236A/ A330K/ (SEQ ID NO: H268D/ I332E 54, 59, 5)S298A GpH7-Kn045/ Kn045 L234Y/ Hl055 G236A/ 60.4 GpH7-Hl055/ L235Y/S239D/ GpL16-k0 G236A/ Q295L/ (SEQ ID NO: H268D/ A330M/ 54, 60, 5) S298AI332E GpH7-Kn056/ Kn056 L234Y/ Hl055 G236A/ 60.4 GpH7-Hl055/ L235Y/S239D/ GpL16-k0 G236A/ Q295L/ (SEQ ID NO: H268D/ A330M/ 55, 60, 5)Q295L/ I332E S298A

The table shows Tm of antibodies with increased binding activities toFcγRIIa and FcγRIIIa.

The result shown in Table 39 demonstrates that all of heterodimerizedantibodies GpH7-Kn045/GpH7-H1048/GpL16-k0,GpH7-Kn045/GpH7-H1055/GpL16-k0, and GpH7-Kn056/GpH7-H1055/GpL16-k0retained high Tm as compared to the homodimerized antibodiesGpH7-Kn037/GpH7-H1036/GpL16-k0 and GpH7-Kn032/GpH7-H1032/GpL16-k0 of theprior art. As described in Example 11, the heterodimerized antibodieshave properties more suitable to achieve FcγR-mediated effector functionas compared to the conventional homodimerized antibodies. Specifically,the finding described above demonstrates that the binding to FcγR can befinely regulated without reducing the physicochemical stability ofantibodies by using the heterodimerized antibody technology.

[Example 14] Effect of a Combination of Alterations that Improve theSelectivity for FcγRIIIa F, an Activating FcγR

As described in Example 8, technologies for improving the selectivityfor activating FcγR and inhibitory FcγR are useful. This Example testswhether the heterodimerization is effective to increase the ratiobetween the binding to FcγRIIIa F, an activating Fcγ, and the binding toFcγRIIb, an inhibitory FcγR, i.e., effective to improve the selectivity,as described in Example 8. Specifically, L234Y, G236W, and S298A (Regiona shown in Table 22-1), which are alterations that increase the ratiobetween the binding to FcγRIIIa F, an activating Fcγ, and the binding toFcγRIIb, an inhibitory FcγR, were combined with S239D, A330L, and I332Eassessed in Example 7, to test whether improvement in selectivity can beachieved in heterodimerized antibodies as compared to homodimerizedantibodies.

To assess this, expression vectors inserted with GpH7-A57 (SEQ ID NO:40) resulting from introduction of all S239D, A330L, and I332E intoGpH7-A5; GpH7-B78 (SEQ ID NO: 41) resulting from introduction of allS239D, A330L, and I332E into GpH7-B3; GpH7-TA7 (SEQ ID NO: 31) resultingfrom introduction of all L234Y, G236W, and S298A into GpH7-A5; andGpH7-TA45 (SEQ ID NO: 32) resulting from introduction of all L234Y,G236W, and S298A into GpH7-B3 were constructed according to the methoddescribed in Reference Example 1. Using these expression vectors andGpH7-A5, GpH7-B3, and GpL16-k0, hetero GpH7-TA7/GpH7-B78/GpL16-k0 one ofwhose H chains had been introduced with L234Y, G236W, and S298A and theother had been introduced with S239D, A330L, and I332E;

GpH7-TA7/GpH7-B3/GpL16-k0 only one of whose H chains had been introducedwith L234Y, G236W, and S298A;

GpH7-TA7/GpH7-TA45/GpL16-k0 both of whose H chains had been introducedwith L234Y, G236W, and S298A;

GpH7-A5/GpH7-B78/GpL16-k0 only one of whose H chains had been introducedwith S239D, A330L, and I332E; and

GpH7-A57/GpH7-B78/GpL16-k0 both of whose H chains had been introducedwith S239D, A330L, and I332E

were expressed and prepared according to the method described inReference Example 1. The prepared antibodies were assayed as to the KDsfor FcγRIIIa and FcγRIIb according to the method described in ReferenceExample 2. Whether each antibody was improved for the FcγRIIIa-bindingselectivity was assessed using, as an indicator, FcγRIIIa/FcγRIIb ratiothat is obtained by dividing KD of each antibody for FcγRIIb by KD ofeach antibody for FcγRIIIa. The assessment result is summarized in Table40.

TABLE 40 FcγRIIIa F/ MUTATION MUTATION FcγRIIIa F FcγRIIb FcγRIIb SAMPLEH1 SITE H2 SITE KD (M) KD (M) ratio GpH7-G1d/GpL16-k0 G1d G1d 1.2E−063.1E−06 2.5 (SEQ ID NO: 2, 5) GpH7-A5/GpH7-B3/ A5 — — — B3 — — — 1.6E−063.1E−06 1.9 GpL16-k0 (SEQ ID NO: 3, 4, 5) GpH7-TA7/GpH7-B3/ TA7 L234YG236W S298A B3 — — — 3.2E−07 4.5E−06 14 GpL16-k0 (SEQ ID NO: 31, 4, 5)GpH7-A5/GpH7-B78/ A5 — — — B78 S239D A330L I332E 5.4E−08 4.3E−07 7.9GpL16-k0 (SEQ ID NO: 3, 41, 5) GpH7-A57/GpH7-B78/ A57 S239D A330L I332EB78 S239D A330L I332E 6.2E−09 6.1E−07 100 GpL16-k0 (SEQ ID NO: 40, 41,5) GpH7-TA7/GpH7-TA45/ TA7 L234Y G236W S298A TA45 L235Y G237W S299A3.3E−06 1.8E−05 5.3 GpL16-k0 (SEQ ID NO: 31, 32, 5) GpH7-TA7/GpH7-B78/TA7 L234Y G236W S298A B78 S239D A330L I332E 4.7E−09 1.1E−06 244 GpL16-k0(SEQ ID NO: 31, 41, 5)

The column “SAMPLE” indicates antibody names; the columns “H1” and “H2”indicate names of the H chain constant region in each antibody; and thecolumn “MUTATION SITE” indicates mutations that are different incomparison with GpH7-A5/GpH7-B3/GpL16-k0 (“-”: used when there is noparticular mutation). Values obtained by dividing the KD of eachantibody for FcγRIIb by the KD of each antibody for FcγRIIIa F areindicated under “FcγRIIIa F/FcγRIIb ratio”. The SEQ ID NOs are alsoshown for the amino acid sequences of the H chain and L chain of eachantibody.

When, based on the results shown in Table 40, a native IgG1GpH7-G1d/GpL16-k0 is compared to GpH7-A5/GpH7-B3/GpL16-k0 in whichD356K, H435R, and K439E each have been introduced into one H chain, theFcγRIIIa/FcγRIIb ratio is 2.5 and 1.9, respectively, and there was nogreat difference between them. This suggests that alterations D356K,H435R, and K439E do not affect the selectivity of the binding toFcγRIIIa.

The effect of each alteration on homodimerized antibodies of prior-arttechnique was verified. The FcγRIIIa/FcγRIIb ratio of homodimerizedantibody GpH7-A57/GpH7-B78/GpL16-k0 both of whose H chains had beenintroduced with S239D, A330L, and I332E was 100, and was increased ascompared to GpH7-A5/GpH7-B3/GpL16-k0. Meanwhile, the FcγRIIIa/FcγRIIbratio of homodimerized antibody GpH7-TA7/GpH7-TA45/GpL16-k0 both ofwhose H chains had been introduced with L234Y, G236W, and S298A was 5.3.Regarding homodimerized antibodies, the combination of S239D, A330L, andI332E was demonstrated to have the greater effect to improve theselectivity of the binding to FcγRIIIa.

Next, heterodimerized antibodies only one of whose H chains had beenintroduced with each alteration group were assessed for the effect ofeach alteration group. The FcγRIIIa/FcγRIIb ratio of heterodimerizedantibody GpH7-A5/GpH7-B78/GpL16-k0 one of whose H chain had beenintroduced with S239D, A330L, and I332E was 7.9, and was increased ascompared to GpH7-A5/GpH7-B3/GpL16-k0. Meanwhile, the FcγRIIIa/FcγRIIbratio of heterodimerized antibody GpH7-TA7/GpH7-B3/GpL16-k0 one of whoseH chain had been introduced with L234Y, G236W, and S298A was 14. Theseresults demonstrate that, regarding heterodimerized antibodies, thegroup of alterations L234Y, G236W, and S298A has the greater effect toincrease the selectivity of the binding to FcγRIIIa.

The difference in the effect of each alteration group in homodimerizedantibodies and heterodimerized antibodies was assessed. Regarding S239D,A330L, and I332E, the FcγRIIIa/FcγRIIb ratio of the heterodimerizedantibody with them was 7.9 while that of the homodimerized antibody withthem was 100. These results demonstrate that S239D, A330L, and I332Ehave the effect to improve the selectivity of the binding to FcγRIIIaupon heterodimerization, and the effect is further enhanced uponhomodimerization. On the other hand, regarding L234Y, G236A, and S298A,the FcγRIIIa/FcγRIIb ratio of the heterodimerized antibody with them was14, while that of the homodimerized antibody with them was 5.3. Theseresults demonstrate that L234Y, G236A, and S298A have the effect toimprove the selectivity of the binding to FcγRIIIa uponheterodimerization, but the effect is reduced upon homodimerization. Theresults demonstrate that the group of alterations S239D, A330L, andI332E in homodimerized antibodies has the greater effect to improve theselectivity of the binding to FcγRIIIa than the group of alterationsL234Y, G236A, and S298A, while the group of alterations L234Y, G236A,and S298A in heterodimerized antibodies has the greater effect toimprove the selectivity of the binding to FcγRIIIa than the group ofalterations S239D, A330L, and I332E.

It was demonstrated that The FcγRIIIa/FcγRIIb ratio of heterodimerizedantibody GpH7-TA7/GpH7-B78/GpL16-k0 with the group of alterations L234Y,G236A, and S298A in combination with the group of alterations S239D,A330L, and I332E was 244, and thus its effect to improve the selectivityof the binding to FcγRIIIa was higher as compared to heterodimerizedantibody GpH7-TA7/GpH7-B3/GpL16-k0 only one of whose H chains had thegroup of alterations L234Y, G236A, and S298A, homodimerized antibodyGpH7-TA7/GpH7-TA45/GpL16-k0 both of whose H chains has the group ofalterations L234Y, G236A, and S298A, heterodimerized antibodyGpH7-A5/GpH7-B78/GpL16-k0 only one of whose H chains has the group ofalterations S239D, A330L, and I332E, and homodimerized antibodyGpH7-A57/GpH7-B78/GpL16-k0 both of whose H chains have the group ofalterations S239D, A330L, and I332E. These results are thought to be asum of the effect of the group of alterations L234Y, G236A, and S298A ina heterodimerized antibody to improve the selectivity of the binding toFcγRIIIa and the effect of the group of alterations S239D, A330L, andI332E in a heterodimerized antibody. Specifically, it was revealed thatheterodimerized antibodies show excellent effect in improving theselectivity of the binding to FcγRIIIa as compared to homodimerizedantibodies.

Specifically, it was shown that the use of heterodimerized antibodies,instead of conventional homodimerized antibodies, enables fineroptimization of the asymmetric interaction between Fc region andFcγRIIIa and design Fc regions having greater selectivity of the bindingto FcγRIIIa.

[Example 15] Measurement of ADCC Activity of Heterodimerized Antibodiesthat Exhibit Enhanced FcgRIIa Binding

The ADCC activity of GpH7-G1d/GpL16-k0, GpH7-Kn033/GpH7-H1033/GpL16-k0,GpH7-Kn037/GpH7-H1036/GpL16-k0, GpH7-Kn032/GpH7-H1032/GpL16-k0,GpH7-Kn045/GpH7-H1048/GpL16-k0, and GpH7-Kn056/GpH7-H1055/GpL16-k0prepared in Example 12 was evaluated according to the method describedin Reference Example 7. The results are summarized in FIG. 33.

When GpH7-G1d/GpL16-k0 and GpH7-Kn033/GpH7-H1033/GpL16-k0 were comparedfor ADCC activity based on FIG. 33, they had comparable ADCC activity.This result demonstrates that knobs-into-holes, even when introducedinto an antibody Fc region, does not affect the FcγR binding or ADCCactivity.

Both heterodimerized antibodies GpH7-Kn045/GpH7-H1048/GpL16-k0 andGpH7-Kn056/GpH7-H1055/GpL16-k0 described in Example 12 exhibited greaterADCC activity as compared to antibody GpH7-Kn033/GpH7-H1033/GpL16-k0before introduction of alteration. Meanwhile, heterodimerized antibodiesGpH7-Kn045/GpH7-H1048/GpL16-k0 and GpH7-Kn056/GpH7-H1055/GpL16-k0 showedADCC activity comparable to that of homodimerized antibodyGpH7-Kn037/GpH7-H1036/GpL16-k0 both of whose H chains have thealteration G236A/S239D/I332E which had been reported to enhance theFcgRIIa R and FcgRIIa H binding and ADCP activity and to that ofantibody GpH7-Kn032/GpH7-H1032/GpL16-k0 resulting from application ofexisting ADCC activity enhancement.

Specifically, as shown in Example 12, GpH7-Kn045/GpH7-H1048/GpL16-k0 andGpH7-Kn056/GpH7-H1055/GpL16-k0 not only exhibit further enhanced bindingto FcgRIIa R and FcgRIIa H as compared to the existing technique butalso have ADCC activity-enhancing effect comparable to the existing ADCCactivity-enhancing technique. Specifically, heterodimerized antibodiesassessed herein are superior to the existing technique in that not onlythey have the ADCC activity-enhancing effect comparable to that achievedby the existing technique but also exhibit enhanced binding to FcgRIIa Hand FcgRIIa R.

[Example 16] Preparation of Heterodimerized AntibodyH240-Kn061/H240-H1071/L73-k0 that Exhibits Enhanced FcgRIIIa Binding

As described in Example 11, heterodimerized antibodies with increasedFcγRIIIa-binding activity were demonstrated to have improved ADCCactivity. In Example 11, the effect was demonstrated with antibodiesagainst GPC3. Whether the same effect can be observed for other antigenswas assessed by conducting a similar experiment using an anti-epiregulin(EREG) antibody. Herein, the H chain variable region sequence of theantibody against EREG is referred to as H240 (SEQ ID NO: 80), and its Lchain sequence including the variable and constant regions is referredto as L73-k0 (SEQ ID NO: 106).

Based on the results of Example 4, new variants having H chains whichexhibit enhanced FcgRIIIa binding were prepared. Herein, theknobs-into-holes technique described in Example 12 was used as theheterodimerization technique. Specifically, H240-Kn033 (SEQ ID NO: 84)resulting from introduction of alterations Y349C and T366W into theconstant region of H240-G1d (SEQ ID NO: 83) and H240-H1033 (SEQ ID NO:85) resulting from introduction of alterations D356C, T366S, L368A, andY407V into the constant region of H240-G1d were prepared according tothe method described in Reference Example 1. Then, H240-Kn061 (SEQ IDNO: 81) was prepared by introducing L234Y, L235Y, G236W, H268D, andS298A into H240-Kn033 (SEQ ID NO: 84) according to the method describedin Reference Example 1. H240-H1071 (SEQ ID NO: 82) was constructed byintroducing K326D, A330M, and K334E into H240-H1033 (SEQ ID NO: 85)according to the method described in Reference Example 1.Heterodimerized antibody H240-Kn061/H240-H1071/L73-k0 was expressed bycombining H240-Kn061, H240-H1071, and L73-k0 according to the methoddescribed in Reference Example 1.

Variants introduced with S239D, A330L, and I332E, which have beenreported to enhance the binding to FcγRIIIa, were constructed in thesame manner as described in Example 12, to be used for comparison.Specifically, H240-Kn032 (SEQ ID NO: 86) and H240-H1032 (SEQ ID NO: 87),resulting from introduction of S239D, A330L, and I332E into H240-Kn033(SEQ ID NO: 84) and H240-H1033 (SEQ ID NO: 85), respectively, wereprepared according to the method described in Reference Example 1.Homodimerized antibody H240-Kn032/H240-H1032/L73-k0 was expressed bycombining H240-Kn032, H240-H1032, and L73-k0 according to the methoddescribed in Reference Example 1.

Then, an afucosylated antibody, which has been reported to enhance thebinding to FcgRIIIa (Glycobiol. Vol. 17 no. 1 pp. 104-118 (2006)), wasprepared for comparison. The fucose transporter function is inhibited incells where the expression of the fucose transporter gene has beenartificially suppressed on both homologous chromosomes. Antibodieslacking fucose can be obtained by using such cells (WO 2006/067913,etc.). Alternatively, antibodies lacking fucose can also be obtained byproducing antibodies in cells with forced expression of beta1,4-N-acetylglucosaminyltransferase III and Golgi alpha-mannosidase II(Ferrara et al., Biotechnol. Bioeng. (2006) 93 (5), 851-861). H240-G1d(SEQ ID NO: 83) and L73-k0 (SEQ ID NO: 106) were expressed incombination, and antibody H240-afucosyl_G1d/L73-k0 (SEQ ID NOs: 83 and106) was obtained by afucosylating H240-G1d/L73-k0 using theabove-described technologies known to those skilled in the art.

Furthermore, H240-Kn033/H240-H1033/L73-k0 was expressed as a control bycombining H240-Kn033 (SEQ ID NO: 84), H240-H1033 (SEQ ID NO: 85), andL73-k0 (SEQ ID NO: 106) according to the method described in ReferenceExample 1.

The binding activity of the antibodies to each FcgR was evaluatedaccording to the method described in Reference Example 8 and the resultsare summarized in Table 41.

TABLE 41 FcgRIa FcgRIIa R FcgRIIa H FcgRIIb FcgRIIIa F FcgRIIIa V SampleKD (M) KD (M) KD (M) KD (M) KD (M) KD (M) H240-G1d/L73-k0 2.3E−108.8E−07 6.6E−07 6.0E−06 1.4E−06 3.1E−07 H240-Kn033/H240-Hl033/L73-k02.5E−10 1.0E−06 9.3E−07 4.1E−06 2.6E−06 3.9E−07H240-Kn032/H240-Hl032/L73-k0 7.3E−11 3.4E−07 6.9E−07 6.2E−07 9.1E−093.1E−09 H240-afucosyl_G1d/L73-k0 4.3E−10 4.9E−07 7.8E−07 2.6E−06 7.8E−086.9E−09 H240-Kn061/H240-Hl071/L73-k0 1.4E−10 3.5E−07 2.8E−07 1.2E−065.1E−09 1.8E−09

The results shown in Table 41 demonstrate that the binding ofheterodimerized antibody H240-Kn061/H240-H1071/L73-k0, in particular, toFcgRIIIa F and FcgRIIIa V was enhanced as compared toH240-Kn033/H240-H1033/L73-k0, Since heterodimerized antibodyH240-Kn061/H240-H1071/L73-k0 is a variant resulting from introduction ofL234Y/L235Y/G236W/H268D/S298A and K326D/A330M/K334E intoH240-Kn033/H240-H1033/L73-k0, it can be said that the binding of theintroduced alterations to FcgR was enhanced.

The results shown in Table 41 demonstrate that the FcgRIIIa F andFcgRIIIa V binding of heterodimerized antibodyH240-Kn061/H240-H1071/L73-k0 was enhanced as compared toH240-afucosyl_G1d/L73-k0 and H240-Kn032/H240-H1032/L73-k0 resulting fromapplication of the existing ADCC activity-enhancing technique. Thisresult demonstrates that the heterodimerized antibody exhibits thestrong effect to enhance the binding to FcgRIIIa as compared to theconventional homodimerized antibody-based ADCC activity-enhancingtechnique and the afucosylation-based ADCC activity-enhancing technique.

In addition, regarding the FcgRIIa binding that is thought to beimportant for ADCP activity enhancement, the FcgRIIa H-binding of theheterodimerized antibody was enhanced compared to the two antibodies,and its FcgRIIa R binding was enhanced relative toH240-afucosyl_G1d/L73-k0 and comparable to that ofH240-Kn032/H240-H1032/L73-k0.

Whether heterodimerized antibody H240-Kn061/H240-H1071/L73-k0 has thefeature of a heterodimerized antibody, which is that it has enhancedFcgR-binding activity compared to homodimerized antibodies comprisingeach H chain therefrom, was assessed. In the heterodimerized antibodyH240-Kn061/H240-H1071/L73-k0, L234Y/L235Y/G236W/H268D/S298A has beenintroduced into H240-Kn061, which is one of the H chains, andK326D/A330M/K334E has been introduced into H240-H1071, which is theother H chain. The heterodimerized antibody was compared tohomodimerized antibodies comprising each H chain therefrom to assesswhether the heterodimerized antibody has stronger binding activity toeach FcgR. Specifically, H240-H1134 (SEQ ID NO: 88) resulting fromintroduction of L234Y/L235Y/G236W/H268D/S298A into H240-H1033 andH240-Kn132 (SEQ ID NO: 89) resulting from introduction ofK326D/A330M/K334E into H240-Kn033 were constructed according to themethod described in Reference Example 1. Using these expression vectors,homodimerized antibody H240-Kn061/H240-H1134/L73-k0 both of whose Hchains have L234Y/L235Y/G236W/H268D/S298A and homodimerized antibodyH240-Kn132/H240-H1071/L73-k0 both of whose H chains haveK326D/A330M/K334E were expressed according to the method described inReference Example 1. The binding to FcgRIIIa F and FcgRIIIa V ofhomodimerized antibodies and heterodimerized antibodyH240-Kn061/H240-H1071/L73-k0 each of whose H chains hasL234Y/L235Y/G236W/H268D/S298A or K326D/A330M/K334E was measuredaccording to the method described in Reference Example 8. The resultsare summarized in Table 42.

TABLE 42 FcgRIIIa F FcgRIIIa V Sample KD (M) KD (M)H240-Kn061/H240-Hl071/L73-k0 5.1E−09 1.8E−09H240-Kn061/H240-Hl134/L73-k0 6.6E−07 8.6E−08H240-Kn132/H240-Hl071/L73-k0 7.7E−08 1.6E−08

The results shown in Table 42 demonstrate that the FcgRIIIa F- andFcgRIIIa V-binding activity of heterodimerized antibodyH240-Kn061/H240-H1071/L73-k0 one of whose H chains hasL234Y/L235Y/G236W/H268D/S298A and the other H chain hasK326D/A330M/K334E is stronger than the activity of homodimerizedantibody H240-Kn061/H240-H1134/L73-k0 both of whose H chains haveL234Y/L235Y/G236W/H268D/S298A and homodimerized antibodyH240-Kn132/H240-H1071/L73-k0 both of whose H chains haveK326D/A330M/K334E. Namely, H240-Kn061/H240-H1071/L73-k0 was demonstratedto have the feature of a heterodimerized antibody, which is that it hasenhanced FcgR-binding activity compared to homodimerized antibodiescomprising each H chain therefrom.

Next, the ADCC activities of H240-Kn033/H240-H1033/L73-k0,H240-Kn032/H240-H1032/L73-k0, H240-afucosyl_G1d/L73-k0, andH240-Kn061/H240-H1071/L73-k0 were compared according to the methoddescribed in Reference Example 9. The results are summarized in FIG. 34.

The results shown in FIG. 34 demonstrate thatH240-Kn061/H240-H1071/L73-k0 exhibits significantly stronger ADCCactivity as compared to H240-Kn033/H240-H1033/L73-k0. Furthermore, theADCC activity was stronger than that of H240-Kn032/H240-H1032/L73-k0 andH240-afucosyl_G1d/L73-k0 resulting from application of the existing ADCCactivity-enhancing technique. Namely, H240-Kn061/H240-H1071/L73-k0 wasdemonstrated to exhibit stronger ADCC activity than that achieved withthe existing ADCC activity-enhancing technique.

[Example 17] Preparation of Further Variants Using HeterodimerizedAntibody H240-Kn061/H240-H1071/L73-k0 as a Template and AssessmentThereof

H240-Kn061/H240-H1071/L73-k0 exhibiting superior ADCC activity wasdiscovered in Example 16 above. To reveal further variants with superioractivity, a total of about 420 types of variants in which amino acids atpositions 231 to 242 (EU numbering) have been substituted with 18 typesof amino acids excluding Cys and original amino acid in each of the Hchains of H240-Kn061 and H240-H1071 were prepared usingH240-Kn061/H240-H1071/L73-k0 as a template according to the methoddescribed in Reference Example 1. The antibodies were assessed for thebinding to each FcgR. Specifically, the KD value of each variant wascalculated for each of FcgRI, FcgRIIa R, FcgRIIa H, FcgRIIb, FcgRIIIa F,and FcgRIIIa V according to the method described in Reference Example 8,and the KD value of H240-Kn061/H240-H1071/L73-k0 for each of FcgRI,FcgRIIa R, FcgRIIa H, FcgRIIb, FcgRIIIa F, and FcgRIIIa V was divided bythe KD value obtained above. The resultant value was defined as relativeKD, and used as an assessment indicator. Specifically, the degree offolds changed in the KD value of each variant for each FcgR when takingthe KD value of H240-Kn061/H240-H1071/L73-k0 as 1 was used as anassessment indicator. When the relative KD is larger, it means that thebinding of the variant to each FcgR is more strongly enhanced ascompared to H240-Kn061/H240-H1071/L73-k0.

Graphs in which relative KDs of respective variants for FcgRI, FcgRIIaR, FcgRIIa H, FcgRIIb, FcgRIIIa F, or FcgRIIIa V is shown on thevertical axis and rank numbers when arranging relative KDs in ascendingorder are shown on the horizontal axis are displayed in FIGS. 35, 36,37, 38, 39, and 40, respectively.

From the analysis result, variants that enhance the binding to any oneof, or several of FcgRIIa R, FcgRIIIa F, and FcgRIIIa V withoutenhancing the FcgRIIb binding relative to H240-Kn061/H240-H1071/L73-k0were discovered. Alterations and H chains introduced with thealterations are summarized in Table 43 (alterations that enhance thebinding to FcgRIIa R and FcgRIIIa without enhancing the binding toFcgRIIb). Alterations in which the relative KD is 1 or less for FcgRIIband the relative KD value is 1.3 or more for any one of, or several ofFcgRIIa R, FcgRIIIa F, and FcgRIIIa V were selected.

TABLE 43 ALTERATION- INTRODUCED H CHAIN ALTERATION FcgRIIa R FcgRIIa HFcgRIIb FcgRIIIa F FcgRIIIa V H240-Hl071 L235F 1.2 1.1 1 1.9 1.6H240-Hl071 V240A 1 1.2 1 1.3 1.7 H240-Hl071 V240F 1.1 0.9 0.9 1.4 1H240-Hl071 F241L 1.2 1.2 0.8 1.3 1.3 H240-Hl071 F241M 1.3 1.1 0.9 1.31.3 H240-Kn061 Y234E 1.4 0.7 0.5 1.9 1.6 H240-Kn061 Y235N 1 0.7 0.7 1.32

The values shown in Table 43 above indicate the relative KD of eachvariant for each FcgR.

The alterations have the effect to enhance the binding to FcgRIIa thatplays an important role in ADCP activity and the binding to FcgRIIIathat plays an important role in ADCC activity without enhancing thebinding to FcgRIIb, an inhibitory FcgR. Thus, a stronger antitumoractivity can be expected, since the introduction of these alterationsincrease ADCC and ADCP activities without enhancing the antibody'simmunosuppressive action.

Furthermore, variants that enhance the binding to FcgRIIa H and FcgRIIaR without reducing the FcgRIIIa binding relative toH240-Kn061/H240-H1071/L73-k0 were discovered. Alterations and H chainsintroduced with the alterations are summarized in Table 44 (alterationsthat enhance the binding to FcgRIIa without reducing the binding toFcgRIIIa). Alterations in which the relative KD is 0.7 or more forFcgRIIIa F and FcgRIIIa V and the relative KD is 1.5 or more for FcgRIIaH and FcgRIIa R were selected.

TABLE 44 ALTERATION- INTRODUCED H CHAIN ALTERATION FcgRIIa R FcgRIIa HFcgRIIb FcgRIIIa F FcgRIIIa V H240-Kn061 A231E 1.7 1.6 1.4 1.8 2H240-Kn061 A231V 1.6 1.6 1.6 1.3 1.5 H240-Kn061 A231Q 1.5 1.5 1.4 1.51.7 H240-Kn061 Y234L 2.2 1.5 3.5 1.1 1.2 H240-Kn061 Y234I 4.1 2.6 7.71.2 1.3 H240-Kn061 S239M 3.2 2 2.2 2.2 2.8 H240-Kn061 S239I 4.4 2.1 3.13 3.4 H240-Kn061 S239L 4.8 2.4 3.4 3.4 3.7 H240-Kn061 S239V 4.2 2.4 3.42.4 2.9 H240-Hl071 F241N 3.8 3.5 1.1 0.8 0.7

The values shown in Table 44 above indicate the relative KD of eachvariant for each FcgR.

The alterations enhance the binding to FcgRIIa that plays an importantrole in ADCP activity without reducing the binding to FcgRIIIa thatplays an important role in ADCC activity, and some of the alterationsenhance the binding to FcgRIIIa. Thus, a stronger antitumor activity canbe expected by introducing the alterations and thus increasing ADCC andADCP activities, or either ADCC or ADCP activity.

Furthermore, variants that reduce the binding to FcgRIIb withoutreducing the binding to FcgRIIIa relative toH240-Kn061/H240-H1071/L73-k0 were discovered. Alterations and H chainsintroduced with the alterations are summarized in Table 45 (alterationsthat reduce the binding to FcgRIIb while maintaining the binding toFcgRIIIa). Alterations in which the relative KD is 1 or more forFcgRIIIa F and FcgRIIIa V and the relative KD is 0.5 or less for FcgRIIbwere selected.

TABLE 45 ALTERATION- INTRODUCED H CHAIN ALTERATION FcgRIIa R FcgRIIa HFcgRIIb FcgRIIIa F FcgRIIIa V H240-Hl071 V240H 0.7 0.8 0.3 1.3 1H240-Kn061 Y234E 1.4 0.7 0.5 1.9 1.6 H240-Kn061 Y235A 0.5 0.5 0.2 1.21.3 H240-Kn061 Y235I 0.7 0.4 0.2 1.3 1.6 H240-Kn061 Y235P 0.6 0.4 0.51.1 1.5 H240-Kn061 Y235Q 0.8 0.4 0.1 1 1.3 H240-Kn061 Y235V 0.5 0.4 0.21.2 1.3

The values shown in Table 45 above indicate the relative KD of eachvariant for each FcgR.

The alterations reduce the binding to FcgRIIb, an inhibitory FcgR,without reducing the binding to FcgRIIIa that plays an important role inADCC activity. Thus, a stronger antitumor activity can be expected,since introduction of the alterations reduce the antibody'simmunosuppressive action without reducing ADCC activity.

Furthermore, since heterodimerized antibody H240-Kn061/H240-H1071/L73-k0has the feature of a heterodimerized antibody which is that it bindsmore strongly to FcgR than homodimerized antibodies comprising each Hchain therefrom, variants obtained by introducing the alterations intoH240-Kn061/H240-H1071/L73-k0 are thought to have the same feature of aheterodimerized antibody.

[Example 18] Alterations that can Substitute the Alterations Introducedinto the Heterodimerized Antibody H240-Kn061/H240-H1071/L73-k0

Based on the results shown in FIGS. 35, 36, 37, 38, 39, and 40 obtainedin Example 17, whether the alterations in the heterodimerized antibodyH240-Kn061/H240-H1071/L73-k0 were substitutable with other alterationswas assessed. Herein, “substitutable alteration” refers to analteration, when introduced, results in the binding to FcgRIIIa F andFcgRIIIa V of 0.7 times or more, and the binding to FcgRIIb of 1.3 timesor less as compared to before introduction of the alteration.

Regarding the variants prepared in Example 17, alterations wereintroduced into antibodies at amino acid positions 231 to 242 (EUnumbering). In the heterodimerized antibodyH240-Kn061/H240-H1071/L73-k0, L234Y/L235Y/G236W/H268D/S298A has beenintroduced into H chain H240-Kn061 and K326D/A330M/K334E has beenintroduced into H chain H240-H1071. Of the alterations introduced into Hchain H240-Kn061, whether L234Y, L235Y, and G236W are substitutable withother amino acids can be assessed based on the results shown in Example17.

The alteration sites include positions 234, 235, and 236 (EU numbering)in H chain H240-Kn061, which is one of the H chains ofH240-Kn061/H240-H1071/L73-k0, but do not include the alteration sitesintroduced into H chain H240-H1071, which is the other H chain. Of thevariants prepared in this experiment, those introduced with alterationsat positions 234, 235, and 236 (EU numbering) in H chain H240-Kn061,those having a binding to FcgRIIIa F and FcgRIIIa V is 0.7 times or moreand a binding to FcgRIIb is 1.3 times or less as compared toH240-Kn061/H240-H1071/L73-k0 are thought to have a comparable orsuperior activity to H240-Kn061/H240-H1071/L73-k0. Thus, substitutionswithout reducing the excellent properties of heterodimerized antibodyH240-Kn061/H240-H1071/L73-k0 were thought to be possible. Regardingalterations that meet the conditions described above, the alterationsand H chains introduced with them are summarized in Table 46(alterations that allow retaining the activity comparable or that conferthe activity superior to that of H240-Kn061/H240-H1071/L73-k0).

TABLE 46 ALTERATION- INTRODUCED ALTERA- H CHAIN TION FcgRIIb FcgRIIIa FFcgRIIIa V H240-Kn061 Y234A 1.2 0.8 0.9 H240-Kn061 Y234E 0.5 1.9 1.6H240-Kn061 Y234G 0.9 0.8 0.9 H240-Kn061 Y234H 1.3 0.8 0.9 H240-Kn061Y234S 1.0 0.9 0.9 H240-Kn061 Y235A 0.2 1.2 1.3 H240-Kn061 Y235E 0.8 2.23.2 H240-Kn061 Y235F 1.2 1.3 1.5 H240-Kn061 Y235I 0.2 1.3 1.6 H240-Kn061Y235L 0.9 1.8 1.5 H240-Kn061 Y235M 0.7 1.7 1.8 H240-Kn061 Y235N 0.7 1.32.0 H240-Kn061 Y235P 0.5 1.1 1.5 H240-Kn061 Y235Q 0.1 1.0 1.3 H240-Kn061Y235T 0.6 0.9 1.1 H240-Kn061 Y235V 0.2 1.2 1.3 H240-Kn061 Y235W 1.3 1.11.3 H240-Kn061 W236Y 0.8 0.8 1.2

In Table 46 shown above, “ALTERATION-INTRODUCED H CHAIN” means to whichH chain of H240-Kn061/H240-H1071/L73-k0 is it substitutable; and in“ALTERATION”, a numeral represents a residue number according to EUnumbering, the first alphabetical letter represents an amino acidcorresponding to an indicated residue number inH240-Kn061/H240-H1071/L73-k0, and the last alphabetical letterrepresents a substitutable amino acid.

From the results, among the alterations introduced into the H chainconstant region of H240-Kn061, substitutable sites and amino acids aresummarized as shown in Table 47 (alteration sites in H240-Kn061, atwhich an alteration can be substituted while retaining the activitycomparable to that of H240-Kn061/H240-H1071/L73-k0, and substitutableamino acids).

TABLE 47 ALTERED POSITION SUBSTITUTABLE AMINO ACID POSITION 234 Y, A, E,G, H, S POSITION 235 Y, A, E, F, I, L, M, N, P, Q, T, V, W POSITION 236W, Y

In Table 47 shown above, “ALTERED POSITION” refers to a residue numberaccording to EU numbering in H240-Kn061; and “SUBSTITUTABLE AMINO ACID”refers to an amino acid, even when substituted at that site with anamino acid shown in this table, allows having the activity comparable tothat of H240-Kn061/H240-H1071/L73-k0, i.e., an amino acid that can besubstituted.

Of the alterations introduced into H240-Kn061/H240-H1071/L73-k0,regarding H268D and S298A of H240-Kn061, and K326D, A330M, and K334E ofH240-H1071, alterations have not been introduced at the correspondingsites in the experiment described in Example 17. Thus, the presence orabsence of substitutable alterations at the sites described above isalso discussed below based on the results shown in Example 4.Specifically, based on the results shown in Example 4, threealterations, which resulted in an He/Con_3aF value of 130 or more andwhich exhibited the strongest effect at the sites, i.e., those thatshowed a 1.3 fold or more increase as compared to before introduction ofalteration in He/Con_3aF as an indicator for the binding to FcgRIIIa Fin a heterodimerized antibody only one of whose H chains had beenintroduced with an alteration, were selected. The results are summarizedin Table 48 (alteration sites of alterations substitutable with thealteration H268D, S298A, K326D, A330M, or K334E ofH240-Kn061/H240-H1071/L73-k0; substitutable amino acids; and resultingFcgRIIIa F-binding activity).

TABLE 48 ALTERED POSITION SUBSTITUTABLE AMINO ACID He/Con_3aF 268 D 195268 E 184 268 A 121 298 A 151 326 T 140 326 D 148 326 I 153 330 P 152330 M 139 330 F 144 334 I 156 334 E 186 334 D 164

In Table 48 shown above, “ALTERED POSITION” refers to a residue numberaccording to EU numbering. “SUBSTITUTABLE AMINO ACID” refers to thethree alterations which results in a 1.3 fold or more increase ascompared to before introduction of alteration in He/Con_3aF as anindicator for the binding to FcgRIIIa F binding, in a heterodimerizedantibody in which an alteration had been introduced into only one Hchain as described in Example 4, and which exhibits the strongest effectat the sites. “He/Con_3aF” is a value defined in Example 4.

Based on the results shown in Table 48, substitutable amino acids ateach alteration site are summarized in Table 49 (substitutablealteration sites of alteration H268D, S298A, K326D, A330M, or K334E inH240-Kn061/H240-H1071/L73-k0 and substitutable amino acids).

TABLE 49 ALTERED POSITION SUBSTITUTABLE AMINO ACID 268 D, E, A 298 A 326T, D, I 330 P, M, F 334 I, E, D

Based on Table 49, even if D of H268D is substituted with E or A inH240-Kn061/H240-H1071/L73-k0, comparable activity is thought to beachieved. Likewise, even when D of K326D is T or I, or even when M ofA330M is P or F, or even when D of K334D is E or I, comparable activityis thought to be achieved. Meanwhile, as to S298A, no alterations thatis thought to achieve a comparable activity could be found.

Furthermore, since heterodimerized antibody H240-Kn061/H240-H1071/L73-k0has the feature of a heterodimerized antibody which is that it enhancesthe binding activity to FcgR than homodimerized antibodies comprisingeach H chain therefrom, variants obtained by introducing the alterationsinto H240-Kn061/H240-H1071/L73-k0 are thought to have the same featureof heterodimerized antibody.

[Example 19] Introduction of D270E into Heterodimerized AntibodyH240-Kn061/H240-H1071/L73-k0 and Assessment Thereof

Then, to further improve H240-Kn061/H240-H1071, the present inventorstried to further enhance the FcgRIIIa binding which is thought toincrease ADCC activity and to further reduce the FcgRIIb binding whichis thought to reduce the antitumor activity of an antibody viaimmunosuppressive signals. Specifically, D270E, an alteration found inExample 4, which enhances the FcgRIIIa binding and reduces the FcgRIIbbinding, was introduced into both H chains ofH240-Kn061/H240-H1071/L73-k0. The sequences obtained by introducingD270E into H240-Kn061 and H240-H1071 were named H240-Kn072 (SEQ ID NO:90) and H240-H1076 (SEQ ID NO: 91), and were combined with L73-k0 toexpress and prepare the heterodimerized antibodyH240-Kn072/H240-H1076/L73-k0 according to the method described inExample 1. Along with this antibody, H240-Kn033/H240-H1033/L73-k0,H240-Kn032/H240-H1032/L73-k0, H240-afucosyl_G1d/L73-k0, andH240-Kn061/H240-H1071/L73-k0 were assayed for the binding activity toeach FcgR according to the method described in Reference Example 8. Theresults are summarized in Table 50.

TABLE 50 FcgRIa FcgRIIa R FcgRIIa H FcgRIIb FcgRIIIa F FcgRIIIa V SampleKD (M) KD (M) KD (M) KD (M) KD (M) KD (M) H240-Kn033/H240-Hl033/L73-k02.5E−10 1.0E−06 9.3E−07 4.2E−06 2.6E−06 3.9E−07H240-Kn032/H240-Hl032/L73-k0 7.3E−11 3.4E−07 6.9E−07 6.3E−07 9.1E−093.1E−09 H240-afucosyl_G1d/L73-k0 4.3E−10 4.9E−07 7.8E−07 2.6E−06 7.9E−086.9E−09 H240-Kn061/H240-Hl071/L73-k0 1.4E−10 3.5E−07 2.8E−07 1.2E−065.1E−09 1.8E−09 H240-Kn072/H240-Hl076/L73-k0 1.0E−10 7.1E−07 1.3E−075.7E−06 2.6E−09 1.0E−09

Table 50 shows that, similarly to H240-Kn061/H240-H1071/L73-k0,H240-Kn072/H240-H1076/L73-k0 more strongly bound to FcgRIIIa F andFcgRIIIa V than H240-Kn032/H240-H1032/L73-k0 andH240-afucosyl_G1d/L73-k0 resulting from application of the existing ADCCactivity-enhancing technique, and in addition it bound more stronglythan H240-Kn061/H240-H1071/L73-k0. The FcgRIIb binding ofH240-Kn072/H240-H1076/L73-k0 was reduced relative to those ofH240-Kn032/H240-H1032/L73-k0 and H240-afucosyl_G1d/L73-k0 produced bythe existing ADCC activity-enhancing technique, and in addition it wasreduced relative to that of H240-Kn061/H240-H1071/L73-k0.

Specifically, since introduction of D270E enhances the FcgRIIIa bindingthat increases ADCC activity, a stronger ADCC activity is expected, andsince the binding to FcgRIIb that transduces immunosuppressive signalsis impaired, reduction in the antibody's immunosuppressive action isexpected. Thus, H240-Kn072/H240-H1076/L73-k0 is thought to exhibitantitumor effect that is superior to that ofH240-Kn061/H240-H1071/L73-k0.

Then, the ADCC activity of H240-Kn072/H240-H1076/L73-k0 was compared tothose of H240-Kn061/H240-H1071/L73-k0, H240-Kn033/H240-H1033/L73-k0, andH240-afucosyl_G1d/L73-k0. The results are shown in FIG. 41.

The results shown in FIG. 41 demonstrate thatH240-Kn072/H240-H1076/L73-k0 exhibits ADCC activity that issignificantly stronger than that of H240-Kn033/H240-H1033/L73-k0.Furthermore, the ADCC activity of H240-Kn072/H240-H1076/L73-k0 wasstronger than that of afucosylated antibody H240-afucosyl_G1d/L73-k0resulting from application of the existing ADCC activity-enhancingtechnique, and was comparable to that of H240-Kn061/H240-H1071/L73-k0.

These results demonstrate that heterodimerized antibodyH240-Kn072/H240-H1076/L73-k0 not only has ADCC activity stronger thanthat achieved by the existing ADCC activity-enhancing technique but alsoexhibits reduced binding to FcgRIIb and thus is a more excellentantibody than those obtained by the existing technique.

Furthermore, since heterodimerized antibody H240-Kn061/H240-H1071/L73-k0has the feature of a heterodimerized antibody which is that it bindsmore strongly to FcgR than homodimerized antibodies comprising each Hchain therefrom, H240-Kn072/H240-H1076/L73-k0 obtained by introducingD270E into both H chains of H240-Kn061/H240-H1071/L73-k0 is thought tohave the same feature of heterodimerized antibody.

[Example 20] Further Improvement of Heterodimerized AntibodyH240-Kn072/H240-H1076/L73-k0

The present inventors tried to further improveH240-Kn072/H240-H1076/L73-k0, which was discovered in Example 19.Specifically, H240-Kn072/H240-H1076/L73-k0 was combined with alterationsY234E, Y235N, Y235Q, and S239M that, when introduced into H240-Kn061,additionally confer superior properties to H240-Kn061/H240-H1071/L73-k0discovered in Example 18.

H240-Kn113 (SEQ ID NO: 92) resulting from introduction of Y234E andY235N into H240-Kn072, H240-Kn115 (SEQ ID NO: 93) resulting fromintroduction of S239M into H240-Kn072, and H240-Kn125 (SEQ ID NO: 94)resulting from introduction of Y235Q and S239M into H240-Kn072 wereprepared according to the method described in Reference Example 1.H240-Kn113/H240-H1076/L73-k0, H240-Kn115/H240-H1076/L73-k0, andH240-Kn125/H240-H1076/L73-k0 were prepared by combining H240-H1076 as anH chain, L73-k0 as the L chain, and H240-Kn113, H240-Kn115, andH240-Kn125 as the other H chain according to the method described inReference Example 1. Those described above were assessed for the bindingto each FcgR according to the method described in Reference Example 8,along with native IgG1 H240-G1d/L73-k0, H240-Kn033/H240-H1033/L73-k0modified therefrom using knobs-into-holes, the afucosylated antibodyH240-afucosyl_G1d/L73-k0 obtained by the existing ADCCactivity-enhancing technique, and the homodimerized antibodyH240-Kn032/H240-H1032/L73-k0 both of whose H chains had been introducedwith an ADCC activity-enhancing alteration S239D/A330L/I332E. Theresults are summarized in Table 51.

TABLE 51 FcgRIa FcgRIIa R FcgRIIa H FcgRIIb FcgRIIIa F FcgRIIIa V SampleKD (M) KD (M) KD (M) KD (M) KD (M) KD (M) H240-G1d/L73-k0 2.3E−108.8E−07 6.6E−07 6.0E−06 1.4E−06 3.1E−07 H240-Kn033/H240-Hl033/L73-k02.5E−10 1.0E−06 9.3E−07 4.2E−06 2.6E−06 3.9E−07H240-Kn032/H240-Hl032/L73-k0 7.3E−11 3.4E−07 6.9E−07 6.3E−07 9.1E−093.1E−09 H240-afucosyl_G1d/L73-k0 4.3E−10 4.9E−07 7.8E−07 2.6E−06 7.9E−086.9E−09 H240-Kn072/H240-Hl076/L73-k0 1.0E−10 7.1E−07 1.3E−07 5.7E−062.6E−09 1.0E−09 H240-Kn113/H240-Hl076/L73-k0 2.1E−10 6.0E−07 3.5E−075.0E−06 1.7E−09 6.2E−10 H240-Kn115/H240-Hl076/L73-k0 2.7E−10 1.3E−076.2E−08 1.3E−06 1.4E−09 4.3E−10 H240-Kn125/H240-Hl076/L73-k0 2.4E−103.8E−07 1.9E−07 4.2E−06 1.2E−09 3.7E−10

When compared to H240-Kn072/H240-H1076/L73-k0,H240-Kn113/H240-H1076/L73-k0 showed comparable binding to FcgRIIb, aninhibitory FcgR, and enhanced binding to FcgRIIIa F and FcgRIIIa V thatplay an important role in ADCC activity. Regarding the binding toFcgRIIb, an inhibitory FcgR, the degree of binding was comparable evenwhen compared to native antibody IgG1. With respect to FcgRIIIa, thebinding to FcgRIIIa F and FcgRIIIa V was more enhanced than afucosylatedantibody H240-afucosyl_G1d/L73-k0 obtained by the existing ADCCactivity-enhancing technique and homodimerized antibodyH240-Kn032/H240-H1032/L73-k0 both of whose H chains had been introducedwith ADCC activity-enhancing alterations S239D/A330L/I332E. The findingsdescribed above suggest the possibility thatH240-Kn113/H240-H1076/L73-k0 does not increase immunosuppressive actionsas compared to native IgG1 and exhibits antitumor effect stronger thanthat of afucosylated antibody resulting from application of the existingADCC activity-enhancing alteration and that of the homodimerizedantibody.

When compared to H240-Kn113/H240-H1076/L73-k0,H240-Kn115/H240-H1076/L73-k0 showed enhanced binding to FcgRIIIa F andFcgRIIIa V, which play an important role in ADCC activity. In addition,the binding to FcgRIIa R and FcgRIIa H, which are important for ADCPactivity, was more enhanced than that of native IgG1 H240-G1d/L73-k0,H240-Kn033/H240-H1033/L73-k0 resulting from application ofknobs-into-holes thereto, the afucosylated antibodyH240-afucosyl_G1d/L73-k0 obtained by the existing ADCCactivity-enhancing technique, and the homodimerized antibodyH240-Kn032/H240-H1032/L73-k0 both of whose H chains had been introducedwith ADCC activity-enhancing alterations S239D/A330L/I332E.

As with H240-Kn113/H240-H1076/L73-k0, H240-Kn125/H240-H1076/L73-k0showed more enhanced binding to FcgRIIIa F and FcgRIIIa V, which play animportant role in ADCC activity, than H240-Kn115/H240-H1076/L73-k0,while retaining the binding to FcgRIIb, an inhibitory FcgR, at acomparable level to IgG1. H240-Kn125/H240-H1076/L73-k0 exhibitedenhanced binding to FcgRIIa H, an FcgRIIa allotype as well as reducedFcgRIIb binding and enhanced binding to both FcgRIIIa allotypes ascompared to the afucosylated antibody H240-afucosyl_G1d/L73-k0 obtainedby the existing ADCC activity-enhancing technique and the homodimerizedantibody H240-Kn032/H240-H1032/L73-k0 both of whose H chains had beenintroduced with an ADCC activity-enhancing alteration S239D/A330L/I332E.Thus, H240-Kn125/H240-H1076/L73-k0 can be expected to increase ADCP andADCC activities and also reduce immunosuppressive actions more stronglythan the homodimerized antibody and afucosylated antibody resulting fromapplication of the existing ADCC activity-enhancing alteration.

Then, the ADCC activities of H240-Kn113/H240-H1076/L73-k0,H240-Kn115/H240-H1076/L73-k0, and H240-Kn125/H240-H1076/L73-k0 werecompared to those of H240-Kn033/H240-H1033/L73-k0 and afucosylatedantibody H240-afucosyl_G1d/L73-k0 obtained by the existing ADCCactivity-enhancing technique. The results are shown in FIG. 42.

As shown in FIG. 42, all the heterodimerized antibodies exhibited moreexcellent ADCC activity than the afucosylated antibody obtained by theexisting ADCC activity-enhancing technique.

The profiles of binding to each FcgR and the result of ADCC activitycomparison with the existing technologies demonstrate that Y234E, Y235N,Y235Q, and S239M, which were introduced into H240-Kn072 ofH240-Kn072/H240-H1076/L73-k0, are alterations that conferH240-Kn072/H240-H1076/L73-k0 with further superior characteristics.

Since heterodimerized antibody H240-Kn061/H240-H1071/L73-k0 has thefeature of a heterodimerized antibody which is that it binds morestrongly to FcgR than homodimerized antibodies comprising each H chaintherefrom, H240-Kn072/H240-H1076/L73-k0 obtained by introducing D270Einto both H chains of H240-Kn061/H240-H1071/L73-k0 is expected to havethe same feature of heterodimerized antibody. Likewise,H240-Kn113/H240-H1076/L73-k0, H240-Kn115/H240-H1076/L73-k0, andH240-Kn125/H240-H1076/L73-k0 obtained by introducing further alterationsinto H240-Kn072/H240-H1076/L73-k0 are also thought to have the samefeature of heterodimerized antibody.

[Example 21] Preparation of Heterodimerized Antibodies that ExhibitEnhanced Binding to FcgRIIa and FcgRIIIa

Based on the result shown in Example 4, variants with H chains thatexhibit enhanced binding to FcgRIIa and FcgRIIIa were prepared.Specifically, H240-Kn067 (SEQ ID NO: 95) was produced by introducingL234Y, L235Y, G236W, H268D, S298A, an A327D into H240-Kn033 (SEQ ID NO:84), and H240-H1068 (SEQ ID NO: 96) was produced by introducing D270E,K326D, A330K, and K334E into H240-H1033 (SEQ ID NO: 85), according tothe method described in Reference Example 1. Heterodimerized antibodyH240-Kn067/H240-H1068/L73-k0 was expressed by combining H240-Kn067,H240-H1068, and L73-k0 according to the method described in ReferenceExample 1.

First, whether heterodimerized antibody H240-Kn067/H240-H1068/L73-k0 hasthe feature of a heterodimerized antibody which is that it enhances thebinding activity to FcgR than the homodimerized antibodies comprisingeach H chain therefrom was assessed. In the heterodimerized antibodyH240-Kn067/H240-H1068/L73-k0, L234Y/L235Y/G236W/H268D/S298A/A327D hasbeen introduced into H240-Kn067 as an H chain andD270E/K326D/A330K/K334E has been introduced into H240-H1068 as the otherH chain. Then, H240-H1136 (SEQ ID NO: 97) resulting from introduction ofL234Y/L235Y/G236W/H268D/S298A/A327D into H240-H1033 and H240-Kn133 (SEQID NO: 98) resulting from introduction of D270E/K326D/A330K/K334E intoH240-Kn033 were constructed according to the method described inReference Example 1. Using these expression vectors, homodimerizedantibody H240-Kn067/H240-H1136/L73-k0 both of whose H chains haveL234Y/L235Y/G236W/H268D/S298A/A327D and homodimerized antibodyH240-Kn113/H240-H1068/L73-k0 both of whose H chains haveD270E/K326D/A330K/K334E were expressed according to the method describedin Reference Example 1. The homodimerized antibodies and heterodimerizedantibody H240-Kn067/H240-H1068/L73-k0 one of whose H chains hasL234Y/L235Y/G236W/H268D/S298A/A327D and the other H chain hasD270E/K326D/A330K/K334E were assayed for the binding to FcgRIIa H,FcgRIIa R, FcgRIIIaF, and FcgRIIIa V according to the method describedin Reference Example 8. The results are summarized in Table 52.

TABLE 52 FcgRIIa R FcgRIIa H FcgRIIIa F FcgRIIIa V Sample KD (M) KD (M)KD (M) KD (M) H240-Kn067/ 4.9E−08 9.4E−08 7.1E−09 1.6E−09 H240-Hl068/L73-k0 H240-Kn067/ 9.5E−07 2.1E−06 9.6E−07 3.4E−07 H240-Hl136/ L73-k0H240-Kn133/ 4.5E−07 1.6E−07 1.2E−07 1.4E−08 H240-Hl068/ L73-k0

Furthermore, based on this variant, H240-Kn067/H240-H1068/L73-k0 wascombined with alterations Y235Q and S239M that, when introduced intoH240-Kn061, further confer H240-Kn061/H240-H1071/L73-k0 with superiorcharacteristics as discovered in Example 18. Specifically, H240-Kn120(SEQ ID NO: 99) resulting from introduction of S239M into H240-Kn067 andH240-Kn126 (SEQ ID NO: 100) resulting from introduction of Y235Q intoH240-Kn120 were prepared according to the method described in ReferenceExample 1. H240-Kn067/H240-H1068/L73-k0, H240-Kn120/H240-H1068/L73-k0,and H240-Kn126/H240-H1068/L73-k0 were prepared by combining H240-H1068as an H chain and L73-k0 as the L chain with H240-Kn067, H240-Kn120, andH240-Kn126 as the other H chain according to the method described inReference Example 1. Those described above were assayed for the bindingto each FcgR according to the method described in Reference Example 8.The results are summarized in Table 53.

TABLE 53 FcgRIa FcgRIIa R FcgRIIa H FcgRIIb FcgRIIIa F FcgRIIIa V SampleKD (M) KD (M) KD (M) KD (M) KD (M) KD (M) H240-Kn033/H240-Hl033/L73-k02.5E−10 1.0E−06 9.3E−07 4.2E−06 2.6E−06 3.9E−07H240-Kn032/H240-Hl032/L73-k0 7.3E−11 3.4E−07 6.9E−07 6.3E−07 9.1E−093.1E−09 H240-Kn037/H240-Hl036/L73-k0 9.8E−11 2.9E−08 4.6E−08 3.8E−072.1E−08 6.8E−09 H240-afucosyl_G1d/L73-k0 4.3E−10 4.9E−07 7.8E−07 2.6E−067.9E−08 6.9E−09 H240-Kn067/H240-Hl068/L73-k0 2.6E−10 4.9E−08 9.4E−083.7E−07 7.1E−09 1.6E−09 H240-Kn120/H240-Hl068/L73-k0 3.7E−10 1.7E−085.9E−08 1.5E−07 2.4E−09 6.6E−10 H240-Kn126/H240-Hl068/L73-k0 4.0E−107.6E−08 1.7E−07 5.2E−07 3.7E−09 6.4E−10

This result demonstrates that H240-Kn067/H240-H1068/L73-k0,H240-Kn120/H240-H1068/L73-k0, and H240-Kn126/H240-H1068/L73-k0 allexhibit comparable or enhanced FcgRIIIa binding as compared to theexisting ADCC activity-enhanced antibodies: H240-afucosyl_G1d/L73-k0 andH240-Kn032/H240-H1032/L73-k0. Furthermore, the binding to FcgRIIa R andFcgRIIa H, which play an important role in ADCP activity, was enhancedas compared to each of the existing technologies. The above-describedfinding suggests that all of the heterodimerized antibodiesH240-Kn067/H240-H1068/L73-k0, H240-Kn120/H240-H1068/L73-k0, andH240-Kn126/H240-H1068/L73-k0 produced herein have comparable or strongerADCC activity and superior ADCP activity relative to those achieved bythe existing technologies.

In particular, H240-Kn120/H240-H1068/L73-k0 exhibited more enhancedbinding to FcgRIIa R and FcgRIIa H even when compared to thehomodimerized antibody H240-Kn037/H240-H1036/L73-k0 both of whose Hchains have alterations G236A/S239D/I332E reported to enhance thebinding to FcgRIIa R and FcgRIIa H as well as to increase ADCP activity.Specifically, H240-Kn120/H240-H1068/L73-k0 exhibits more enhancedbinding to FcgRIIIa F and FcgRIIIa V than antibodies obtained by theexisting ADCC activity-enhancing technique, and exhibits more enhancedbinding to FcgRIIa R and FcgRIIa H than antibodies obtained by theexisting ADCP activity-enhancing technique. Thus,H240-Kn120/H240-H1068/L73-k0 is an antibody that can have stronger ADCCand ADCP activities than antibodies obtained by the existingtechnologies.

Next, according to Reference Example 9, the ADCC activity of eachvariant was compared to that of the afucosylated antibodyH240-afucosyl_G1d/L73-k0 obtained by the existing ADCCactivity-enhancing technique. The result is shown in FIG. 43.

The result shown in FIG. 43 demonstrates that all of the heterodimerizedantibodies produced this time have ADCC activity comparable or superiorto that of the afucosylated antibody obtained by the existing ADCCactivity-enhancing technique.

The profiles of binding to each FcgR and the result of ADCC activitycomparison with the existing technologies demonstrate that all ofH240-Kn067/H240-H1068/L73-k0, H240-Kn120/H240-H1068/L73-k0, andH240-Kn126/H240-H1068/L73-k0 prepared this time are heterodimerizedantibodies that are highly probable to have ADCC activity comparable orsuperior to that achieved with the existing ADCC activity-enhancingtechnique as well as increased ADCP activity via the FcgRIIa binding.

[Example 22] Assessment of Heterodimerized AntibodyH240-AK072/H240-BH076/L73-k0 for the Binding Activity to Each FcgR andADCC Activity

Hereinabove, H240-Kn072/H240-H1076/L73-k0, which is a heterodimerizedantibody resulting from application of knobs-into-holes, was assessedfor its binding to each FcgR and ADCC activity. Herein, whether thefeature of a heterodimerized antibody which is that a heterodimerizedantibody comprising two different H chains has increased FcgR-bindingactivity than the homodimerized antibodies comprising each H chaintherefrom is also observed even when using D356K, H435R, and K439E as analternative heterodimerized antibody-producing technique, was assessed.

First, H240-AK072 (SEQ ID NO: 104) was constructed by introducingL234Y/L235Y/G236W/H268D/D270E/S298A, an alteration introduced intoH240-Kn072 that is an H chain of H240-Kn072/H240-H1076/L73-k0, intoH240-A5E (SEQ ID NO: 102) resulting from introduction of D356K and H435Rinto H240-G1dE (SEQ ID NO: 101), which is an allotype of H240-G1d (SEQID NO: 83). Then, H240-BH076 (SEQ ID NO: 105) was constructed byintroducing D270E/K326D/A330M/K334E, which was introduced intoH240-H1076 as the other H chain, into H240-B3E (SEQ ID NO: 103)resulting from introduction of K439E into H240-G1dE. The heterodimerizedantibody H240-AK072/H240-BH076/L73-k0 was expressed and prepared bycombining H240-AK072, H240-BH076, and L73-k0 according to the methoddescribed in Example 1. Similarly, H240-AK072 and L73-k0, and H240-BH076and L73-k0 were each combined to express and prepare the homodimerizedantibodies H240-AK072/L73-k0 and H240-BH076/L73-k0. The antibodies werecompared for the binding to each FcgR according to the method describedin Reference Example 8. The results are summarized in Table 54.

TABLE 54 FcgRIIIa F FcgRIIIa V Sample KD (M) KD (M) H240-AK072/L73-k03.0E−07 2.9E−08 H240-BH076/L73-k0 2.6E−08 5.3E−09H240-AK072/H240-BH076/L73-k0 3.7E−09 9.8E−10

The results described above confirm that the heterodimerized antibodyH240-AK072/H240-BH076/L73-k0 has the feature of a heterodimerizedantibody which is that it binds to FcgR more strongly than homodimericantibodies H240-AK072/L73-k0 and H240-BH076/L73-k0 each of whichcomprises either H chain therefrom.

Then, H240-A5E, H240-B3E, and L73-k0 were combined and expressed toprepare H240-A5E/H240-B3E/L73-k0 according to the method described inReference Example 1. H240-G1d/L73-k0, H240-A5E/H240-B3E/L73-k0,H240-afucosyl_G1d/L73-k0, and the heterodimerized antibodyH240-AK072/H240-BH076/L73-k0 were assessed for the binding to each FcgRaccording to the method described in Reference Example 8. The resultsare summarized in Table 55.

TABLE 55 FcgRIa FcgRIIa R FcgRIIa H FcgRIIb FcgRIIIa F FcgRIIIa V SampleKD (M) KD (M) KD (M) KD (M) KD (M) KD (M) H240-G1d/L73-k0 2.3E−108.8E−07 6.6E−07 6.1E−06 1.4E−06 3.1E−07 H240-A5E/H240-B3E/L73-k0 6.1E−109.4E−07 9.1E−07 4.3E−06 1.6E−06 3.3E−07 H240-afucosyl_G1d/L73-k0 4.3E−104.9E−07 7.8E−07 2.6E−06 7.9E−08 6.9E−09 H240-AK072/H240-BH076/L73-k02.4E−10 4.6E−07 1.3E−07 2.9E−06 3.7E−09 9.8E−10

The results show that the binding of H240-AK072/H240-BH076/L73-k0 toFcgRIIIa F and FcgRIIIa V was enhanced as compared to H240-G1d/L73-k0and afucosyl antibody H240-afucosyl_G1d/L73-k0 obtained with theexisting ADCC activity-enhancing technique. Meanwhile, since theFcgR-binding activity was comparable between H240-G1d/L73-k0 andH240-A5E/H240-B3E/L73-k0, the increased binding activity ofH240-AK072/H240-BH076/L73-k0 was thought to be due to alterationsL234Y/L235Y/G236W/H268D/D270E/S298A and D270E/K326D/A330M/K334Eintroduced into the each of H chains.

[Example 23] X-Ray Crystal Structure Analysis of a Complex of Fc(Kn120/H1068) and FcgRIIb Extracellular Region

H240-Kn120/H240-H1068/L73-k0 produced as described in Example 21 notonly had increased binding activity to FcgRIIIa and FcgRIIa type H andto FcgRIIa R allotype but also had increased binding activity toFcgRIIb, an inhibitory receptor. The enhanced FcgRIIb binding is thoughtto cause immunosuppressive effects. Thus, reducing the binding toFcgRIIb would be able to achieve further enhancement of ADCC activity,which is an objective of the present invention.

However, 93% of the amino acid sequence of the extracellular region ofFcgRIIa and FcgRIIb match, and are highly homologous to each other.Furthermore, according to an analysis based on the crystallographicstructure (J. Immunol. 2011, 187, 3208-3217) of the complex between Fcof native IgG1 (hereinafter abbreviated as Fc (WT)) and theextracellular region of FcgRIIa type R, near interacting interfacesbetween the both proteins, only three amino acids (Gln127, Leu132, andPhe160) in FcgRIIa type R were different when compared to FcgRIIb (FIG.44). For this reason, it was predicted that it was extremely difficultto reduce the FcgRIIb-binding activity alone while retaining the bindingactivity to FcgRIIa type R. Then, the present inventors conceived that,if they obtained information about the crystallographic structure of thecomplex between the Fc portion (Fc (Kn120/H1068)) ofH240-Kn120/H240-H1068/L73-k0 and FcgRIIb extracellular region andinvestigated the amino acid mutations to be introduced in more detail,the possibility of revealing an alteration that selectively reduces theFcgRIIb-binding activity would be increased. Thus, the complex betweenFc (Kn120/H1068) and FcgRIIb extracellular region was analyzed by X-raycrystal structure analysis.

As a result, the present inventors succeeded in determining thethree-dimensional structure of the complex of Fc (Kn120/H1068)/FcgRIIbextracellular region at 2.78 Å resolution by X-ray crystal structureanalysis. The structure obtained as a result of the analysis is shown inFIG. 45. FcgRIIb extracellular region was revealed to be bound andsandwiched between two Fc CH2 domains, which resembles thethree-dimensional structures of the previously analyzed complexesbetween Fc (WT) and the respective extracellular regions of FcgRIIIa(Proc. Natl. Acad. Sci. USA, 2011, 108, 12669-126674), FcgRIIIb (Nature,2000, 400, 267-273; J. Biol. Chem. 2011, 276, 16469-16477), and FcgRIIa.

Next, the structures around three amino acid residues, which are nearthe binding interface to Fc and are different between FcgRIIa type R andFcgRIIb, are described. FIG. 46 shows the structure around Lys 127 (Glnin FcgRIIa type R). The closest residue in FcgRIIb is Ala at position298 (EU numbering) located in CH2 domain B of Fc shown in FIG. 46. Thisresidue is in direct contact with FcgRIIb at the binding interface.Therefore, it was thought that introduction of any bulky residue capableof interacting with Lys 127 was difficult. Other surrounding amino acidresidues are too distant from Lys127, and no mutations capable of directinteraction could be found. FIG. 47 shows the structure around Ser132(Leu in FcgRIIa type R). This residue is also too distant from Fc, andno mutations capable of direct interaction with this Ser could be found.Finally, FIG. 48 shows the structure around Tyr160 (Phe in FcgRIIa typeR). This Tyr forms a hydrogen bond with the main chain carbonyl oxygenof Gly at position 236 (EU numbering) in CH2 domain A of Fc. In thiscase, the FcgRIIb-binding activity alone may be reduced by introducing amutation at Gly236 at position 236 (EU numbering), if the mutationcauses a change of the loop structure and thereby eliminates thehydrogen bond. Further, when a mutation with any bulky side chain isintroduced at the position of Gly at position 236 (EU numbering), theside chain is predicted to interact directly with the side chain atposition 160 in FcgRIIa or FcgRIIb, and the FcgRIIb binding activity maybe selectively reduced by using the difference between Phe160 in FcgR2atype R and Tyr160 in FcgRIIb.

Experimental Method

[Expression and Purification of Fc (Kn0120/H1068)]

An Fc (Kn0120/H1068) was prepared as follows. First, Cys at position 220(EU numbering) of H240-Kn120 (SEQ ID NO: 99) and H240-H1068 (SEQ ID NO:96) was substituted with Ser. Then, genetic sequence of Fc (Kn0120) andFc (H1068) from Glu at position 236 (EU numbering) to its C terminal wascloned by PCR. Using this cloned genetic sequence, production ofexpression vectors, and expression and purification of Fc (Kn0120) andFc (H1068) were carried out according to the method of ReferenceExample 1. Cys at position 220 (EU numbering) forms a disulfide bondwith Cys of the L chain in general IgG1. The L chain is not co-expressedwhen Fc alone is prepared, and therefore, this residue was substitutedwith Ser to avoid formation of unnecessary disulfide bonds.

[Expression and Purification of the FcgRIIb Extracellular Region]

This was prepared according to the method of Reference Example 8.

[Purification of the Fc (Kn120/H1068)/FcgRIIb Extracellular RegionComplex]

To 1.5 mg of the FcgRIIb extracellular region sample obtained forcrystallization, 0.15 mg of Endo Fl (Protein Science 1996, 5: 2617-2622)expressed and purified from Escherichia coli as a glutathioneS-transferase fusion protein was added. This was allowed to remain atroom temperature for three days in 0.1 M Bis-Tris buffer at pH 6.5, andthe N-linked oligosaccharide was cleaved, leaving N-acetylglucosaminedirectly bound to Asn. Next, this FcgRIIb extracellular region samplesubjected to carbohydrate cleavage treatment was concentrated byultrafiltration with 10000 MWCO, and purified by gel filtrationchromatography (Superdex200 10/300) using a column equilibrated in 20 mMHEPES at pH 7.5 containing 0.1 M NaCl. Furthermore, to the obtainedcarbohydrate-cleaved FcγRIIb extracellular region fraction,Fc(Kn0120/H1068) was added so that the molar ratio of the FcgRIIbextracellular region would be present in slight excess, and afterconcentration by ultrafiltration with 10,000 MWCO, a sample of the Fc(Kn0120/H1068)/FcgRIIb extracellular region complex was obtained throughpurification by gel filtration chromatography (Superdex200 10/300) usinga column equilibrated in 25 mM HEPES at pH 7.5 containing 0.1 M NaCl.

[Crystallization of the Fc (Kn120/H1068)/FcgRIIb Complex ExtracellularRegion Complex]

A sample of the Fc (Kn120/H1068)/FcgRIIb extracellular region complexwas concentrated to approximately 10 mg/mL by ultrafiltration with10,000 MWCO, and crystallization was carried out by the hanging dropvapor diffusion method in combination with seeding method. VDXm plate(Hampton Research) was used for crystallization. Using a reservoirsolution containing 0.1 M Bis-Tris (pH 6.0), 14.4% PEG3350, and 0.2 Mammonium sulfate, crystallization drops were prepared at a mixing ratioof reservoir solution:crystallization sample=0.85 μl:0.85 μl. Then,streak seeding was performed using Seeding Tool (Hampton Research) totransfer seed crystals from the crystals of the complex obtained underthe same condition. The drops were allowed to stand at 20° C. Thissuccessfully yielded columnar crystals.

[Measurement of X-Ray Diffraction Data from an Fc (Kn120/H1068)/FcgRIIbExtracellular Region Complex Crystal]

One of the obtained single crystals of the Fc (Kn120/H1068)/FcgRIIbextracellular region complex was soaked into a solution of 0.1 MBis-Tris pH 6.0, 17.5% PEG3350, 0.2 M ammonium sulfate, glycerol 16%(v/v). The crystal was fished out of the solution using a pin withattached tiny nylon loop, and frozen in liquid nitrogen; and then X-raydiffraction data was measured at synchrotron radiation facility PhotonFactory BL-17A in High Energy Accelerator Research Organization. Duringthe measurement, the crystal was constantly placed in a nitrogen streamat −178° C. to maintain in a frozen state, and a total of 360 X raydiffraction images were collected using Quantum 315r CCD detector (ADSC)attached to a beam line with rotating the crystal 0.5° at a time.Determination of cell parameters, indexing of diffraction spots, anddiffraction data processing from the obtained diffraction images wereperformed using the Xia2 program (J. Appl. Cryst. 2010, 43: 186-190),XDS Package (Acta. Cryst. 2010, D66: 125-132) and Scala (Acta. Cryst.2006, D62: 72-82); and finally, diffraction intensity data up to 2.78 Åresolution was obtained. The crystal belongs to the space group P4₁2₁2,and has the following cell parameters; a=152.94 Å, b=152.94 Å, c=82.24Å, α=90°, β=90°, γ=90°.

[X Ray Crystallographic Analysis of the Fc (Kn120/H1068)/FcgRIIbExtracellular Region Complex]

Crystal structure of the Fc (Kn120/H1068)/FcgRIIb extracellular regioncomplex was determined by the molecular replacement method using theprogram Phaser ((J. Appl. Cryst. 2007, 40: 658-674). From the size ofthe obtained crystal lattice and the molecular weight of the Fc(Kn120/H1068)/FcgRIIb extracellular region complex, the number ofcomplexes in the asymmetric unit was predicted to be one. From thestructural coordinates of PDB code: 3SGJ which is the crystal structureof the Fc(WT)/FcgRIIIa extracellular region complex, the amino acidresidue portions of the A chain positions 239-340 and the B chainpositions 239-340 were taken out as separate coordinates, and they wereused respectively as models for searching the Fc CH2 domains. The aminoacid residue portions of the A chain positions 341-444 and the B chainpositions 341-443 were taken out as a single set of coordinates from thesame structural coordinates of PDB code: 3SGJ; and this was used as amodel for searching the Fc CH3 domains. Finally, from the structuralcoordinates of PDB code: 2FCB which is a crystal structure of theFcgRIIb extracellular region, the amino acid residue portions of the Achain positions 6-178 was taken out and used as a model for searchingthe FcgRIIb. The orientation and position of each search model in thecrystal lattice were determined for Fc CH3 domain, FcgRIIb extracellularregion, and Fc CH2 domain, based on the rotation function andtranslation function to obtain the initial model for the crystalstructure of the Fc (Kn120/H1068)/FcgRIIb extracellular region complex.When rigid body refinement which moves the two Fc CH2 domains, the twoFc CH3 domains, and the FcgRIIb extracellular region was performed onthe obtained initial model, the crystallographic reliability factor, Rvalue became 41.4%, and the Free R value became 42.6% to diffractionintensity data from 25 Å to 3.0 Å at this point. Furthermore, structuralrefinement using the program REFMACS (Acta Cryst. 2011, D67, 355-367),and revision of the model to observe the electron density maps whosecoefficient have 2Fo-Fc or Fo-Fc, which are calculated based on theexperimentally determined structural factor Fo, the calculatedstructural factor Fc and the calculated phase using the model, wascarried out by the Coot program (Acta Cryst. 2010, D66: 486-501), andmodel refinement was carried out by repeating these steps. Finally, as aresult of incorporation of water molecules into the model based on theelectron density maps which use 2Fo-Fc or Fo-Fc as the coefficient, andthe following refinement, the crystallographic reliability factor, Rvalues and the Free R value of the model containing 4964 non-hydrogenatoms became 22.8% and 27.7% to 24274 diffraction intensity data from 25Å to 2.78 Å resolution, respectively.

[Example 24] Preparation of Antibodies Retaining the FcgRIIaR-BindingActivity or Having Increased FcgRIIaR-Binding Activity, and HavingReduced FcgRIIb-Binding Activity

Variant H240-Kn120/H240-H1068/L73-k0 discovered in Example 21 hadincreased binding activity to FcgRIIaR and FcgRIIaH that are importantfor ADCP activity, and in addition, its binding activity to inhibitoryFcgRIIb was increased to about 50 times that of native IgG1. In order toachieve high ADCP activity, it is preferable to reduce the bindingactivity to inhibitory FcgRIIb as much as possible. Thus, the presentinventors searched for alterations that enable reduction of theFcgRIIb-binding activity while retaining the binding activity toactivating FcgRIIaR and FcgRIIaH. As described in Example 23, the resultof crystallographic analysis of the complex between Fc ofH240-Kn120/H240-H1068/L73-k0 and FcgRIIb extracellular region showedthat Tyr160 in FcgRIIb formed a hydrogen bond with the main chaincarbonyl oxygen of Gly236 present in CH2 domain A of Fc (Kn120/H1068).In FcgRIIaR and FcgRIIaH, the corresponding position is occupied byPhe160, and the above-described interaction is unlikely to be present.This suggests the possibility that, if the interaction to Tyr160 inFcgRIIb can be eliminated by introducing an alteration at Gly236, theFcgRIIb-binding activity may be reduced while retaining the bindingactivity to FcgRIIa type R, i.e., the FcgRIIb-binding activity can beselectively reduced. Specifically, the present inventors conceived thatthe interaction to Y160 could be eliminated if reversion of G236 andalteration of the loop structure was introduced by substituting Ser,Val, Ile, or Thr for G236 in the H chain derived from H240-H1068 thatinteracts with Y160 of FcgRIIb in the binding between FcgRIIb andH240-Kn120/H240-H1068/L73-k0. Meanwhile, though distant, Lys127 ofFcgRIIb may be in electrostatic interaction with E294 of CH2 domain A ofFc (Kn120/H1068). Thus, the present inventors considered the possibilitythat the interaction with FcgRIIb may be reduced by inducingelectrostatic repulsion by substituting E294 with positively charged Lysor Arg.

H240-Kn179 (SEQ ID NO: 107) and H240-Kn180 (SEQ ID NO: 108) resultingfrom introduction of each of E294R and E294K into H240-Kn120 (SEQ ID NO:99), and H240-H1073 (SEQ ID NO: 109), H240-H1085 (SEQ ID NO: 110),H240-H1086 (SEQ ID NO: 111), and H240-H1089 (SEQ ID NO: 112) resultingfrom introduction of each of G236S, G236V, G236I, and G236T intoH240-H1068 (SEQ ID NO: 96), were prepared according to the methoddescribed in Reference Example 1. H240-Kn120/H240-H1073/L73-k0,H240-Kn120/H240-H1085/L73-k0, H240-Kn120/H240-H1086/L73-k0,H240-Kn120/H240-H1089/L73-k0, H240-Kn179/H240-H1068/L73-k0, andH240-Kn180/H240-H1068/L73-k0 were prepared by combining each ofH240-Kn120 and H240-Kn180 as an H chain, L73-k0 as the L chain, andH240-H1073, H240-H1085, H240-H1086, and H240-H1089 as the other H chainaccording to the method described in Reference Example 1. The variantsdescribed above were assessed for their binding to FcgR according to themethod described in Reference Example 8. The results are shown in Table56. The KD of H240-Kn120/H240-H1073/L73-k0 in the FcgRIa column of thetable was calculated under the assumption that kd is 5×10−5 s−1 or less,because the value of kd for FcgRIa was smaller than the measurementlimit 5×10−5 s−1 out of the measurable range of 5×10−5 s−1 to 1 s−1 fordissociation constant (kd) in Biacore4000 used in the assay.

TABLE 56 FcgRIa FcgRIIaR FcgRIIaH FcgRIIb FcgRIIIaF FcgRIIIaV Sample KD(M) KD (M) KD (M) KD (M) KD (M) KD (M) H240-Kn120/H240-HI068/L73-k01.6E−10 2.0E−08 4.1E−08 1.5E−07 2.2E−09 3.6E−10H240-Kn120/H240-HI073/L73-k0 <2.8E−11   1.5E−08 2.1E−08 2.1E−07 8.2E−091.8E−09 H240-Kn120/H240-HI085/L73-k0 8.7E−08 1.9E−08 4.6E−08 3.9E−072.3E−08 6.2E−09 H240-Kn120/H240-HI086/L73-k0 1.4E−09 2.2E−08 5.3E−084.8E−07 3.3E−08 1.4E−08 H240-Kn120/H240-HI089/L73-k0 1.8E−10 1.4E−084.1E−08 1.9E−07 1.2E−08 5.4E−09 H240-Kn179/H240-HI068/L73-k0 3.0E−102.6E−08 4.1E−08 2.6E−07 4.8E−09 9.8E−10 H240-Kn180/H240-HI068/L73-k03.0E−10 2.1E−08 3.3E−08 2.4E−07 3.1E−09 6.7E−10

Furthermore, values obtained by dividing KD ofH240-Kn120/H240-H1068/L73-k0 for each of FcgRIIaR, FcgRIIaH, and FcgRIIbby KD of each variant, the relative KD determined when taking, as 1, theKD of H240-Kn120/H240-H1068/L73-k0 for FcgRIIaR, FcgRIIaH, or FcgRIIbare shown in Table 57.

TABLE 57 FcgRIIaR FcgRIIaH FcgRIIb FcgRIIIaF FcgRIIIaV Relative RelativeRelative Relative Relative sample KD KD KD KD KD H240-Kn120/ 1.0 1.0 1.01.0 1.0 H240-Hl068/ L73-k0 H240-Kn120/ 1.3 2.0 0.7 0.3 0.2 H240-Hl073/L73-k0 H240-Kn120/ 1.1 0.9 0.4 0.1 0.1 H240-Hl085/ L73-k0 H240-Kn120/0.9 0.8 0.3 0.1 0.0 H240-Hl086/ L73-k0 H240-Kn120/ 1.4 1.0 0.8 0.2 0.1H240-Hl089/ L73-k0 H240-Kn179/ 0.8 1.0 0.6 0.5 0.4 H240-Hl068/ L73-k0H240-Kn180/ 1.0 1.2 0.6 0.7 0.5 H240-Hl068/ L73-k0

The results demonstrate that all of the six types of alterationsproduced for this assessment retained or increased the bindingactivities to FcgRIIaR and FcgRIIaH and reduced the FcgRIIb-bindingactivity as compared to H240-Kn120/H240-H1068/L73-k0.

Next, the alterations examined in Table 57 were combined to furtherreduce the FcgRIIb-binding activity. In this experiment, the presentinventors aimed to further suppress the FcgRIIb-binding activity by thecombined use of introduction of E294K or E294R into H240-Kn120 andintroduction of G236T into H240-H1068. As shown in Table 57, all thealterations reduce the binding activities to FcgRIIIa F and FcgRIIIa V.Then, in addition to these alterations, I332E that has been reported toincrease the FcgRIIIa-binding activity and the alteration Y235N thatincreases the FcgRIIIa-binding activity as described in Example 18 wereintroduced, in an aim to further suppress the FcgRIIb-binding activityand increase the FcgRIIIa-binding activity.

H240-Kn192 (SEQ ID NO: 113) resulting from introduction of Y235N andE294K into H240-Kn120 (SEQ ID NO: 99), H240-Kn193 (SEQ ID NO: 114)resulting from introduction of Y235N and E294R into H240-Kn120 (SEQ IDNO: 99), and H240-H1204 (SEQ ID NO: 115) resulting from introduction ofG236T and I332E into H240-H1068 (SEQ ID NO: 96) were prepared accordingto the method described in Reference Example 1.H240-Kn179/H240-H1089/L73-k0, H240-Kn180/H240-H1089/L73-k0,H240-Kn192/H240-H1204/L73-k0, and H240-Kn193/H240-H1204/L73-k0 wereprepared by combining each of H240-Kn192 and H240-Kn193 as an H chain,L73-k0 as the L chain, and H240-H1089 and H240-H1204 as the other Hchain according to the method described in Reference Example 1. Thevariants described above were assessed for their binding to FcγRaccording to the method described in Reference Example 8. The result isshown in Table 58. The KDs of H240-Kn192/H240-H1204/L73-k0 andH240-Kn193/H240-H1204/L73-k0 shown in the FcgRIa column of the tablewere calculated under the assumption that kd is 5×10⁻⁵ s⁻¹ or less,because the value of kd for FcγRIa was smaller than the measurementlimit 5×10⁻⁵ s⁻¹ out of the measurable range of 5×10⁻⁵ s⁻¹ to 1 s⁻¹ fordissociation constant (kd) in Biacore4000 used in the assay.

TABLE 58 FcgRIa FcgRIIaR FcgRIIaH FcgRIIIb FcgRIIIaF FcgRIIIaV Sample KD(M) KD (M) KD (M) KD (M) KD (M) KD (M) H240-Kn120/H240-HI068/L73-k01.6E−10 2.0E−08 4.1E−08 1.5E−07 2.2E−09 3.6E−10H240-Kn120/H240-HI089/L73-k0 1.8E−10 1.4E−08 4.1E−08 1.9E−07 1.2E−085.4E−09 H240-Kn179/H240-HI068/L73-k0 3.0E−10 2.6E−08 4.1E−08 2.6E−074.8E−09 9.8E−10 H240-Kn180/H240-HI068/L73-k0 3.0E−10 2.1E−08 3.3E−082.4E−07 3.1E−09 6.7E−10 H240-Kn179/H240-HI089/L73-k0 4.6E−10 1.8E−084.7E−08   4E−07 3.9E−08 1.8E−08 H240-Kn180/H240-HI089/L73-k0 2.7E−101.6E−08 3.8E−08 3.8E−07 2.4E−08 1.4E−08 H240-Kn192/H240-HI204/L73-k0<1.9E−11   1.9E−08 4.2E−08 2.9E−07 1.2E−08 2.2E−09H240-Kn193/H240-HI204/L73-k0 <2.4E−11   1.6E−08 3.5E−08 2.9E−07 1.1E−081.8E−09

Furthermore, values obtained by dividing KD ofH240-Kn120/H240-H1068/L73-k0 for each of FcgRIIaR, FcgRIIaH, and FcgRIIbby KD of each variant, the relative KD determined when taking, as 1, theKD of H240-Kn120/H240-H1068/L73-k0 for FcgRIIaR, FcgRIIaH, or FcgRIIbare shown in Table 59.

TABLE 59 FcgRIIaR FcgRIIaH FcgRIIb FcgRIIIaF FcgRIIIaV Relative RelativeRelative Relative Relative sample KD KD KD KD KD H240-Kn120/ 1.0 1.0 1.01.0 1.0 H240-Hl068/ L73-k0 H240-Kn120/ 1.4 1.0 0.8 0.2 0.1 H240-Hl089/L73-k0 H240-Kn179/ 0.8 1.0 0.6 0.5 0.4 H240-Hl068/ L73-k0 H240-Kn180/1.0 1.2 0.6 0.7 0.5 H240-Hl068/ L73-k0 H240-Kn179/ 1.1 0.9 0.4 0.1 0.0H240-Hl089/ L73-k0 H240-Kn180/ 1.3 1.1 0.4 0.1 0.0 H240-Hl089/ L73-k0H240-Kn192/ 1.1 1.0 0.5 0.2 0.2 H240-Hl204/ L73-k0 H240-Kn193/ 1.3 1.20.5 0.2 0.2 H240-Hl204/ L73-k0

H240-Kn179/H240-H1089/L73-k0 and H240-Kn180/H240-H1089/L73-k0, resultingfrom introduction of E294K or E294R into H240-Kn120 and introduction ofG236T into H240-H1068, both exhibited enhanced binding to FcgRIIaR andFcgRIIaH as compared to H240-Kn120/H240-H1068/L73-k0, while theirFcgRIIb binding was reduced to 0.4 times that ofH240-Kn120/H240-H1068/L73-k0. Furthermore, the effect to reduce theFcgRIIb binding was 1.5 times to twice when compared toH240-Kn120/H240-H1089/L73-k0, H240-Kn179/H240-H1068/L73-k0, andH240-Kn180/H240-H1068/L73-k0, only one of whose chains has eachalteration.

H240-Kn192/H240-H1204/L73-k0 and H240-Kn193/H240-H1204/L73-k0 resultingfrom introduction of I332E and Y235N into the above variants bothretained the binding to FcgRIIaR, FcgRIIaH, and FcgRIIb and enhanced theFcgRIIIaF and FcgRIIIaV binding to twice and 8 times, respectively,compared to H240-Kn179/H240-H1089/L73-k0 andH240-Kn180/H240-H1089/L73-k0 before introduction of the alterations.

[Example 25] Enhancement of the Binding Activity of the HeterodimerizedAntibody H240-Kn120/H240-H1068/L73-k0 to Activating FcgR

In Example 24, variants having reduced binding activity to inhibitoryFcgRIIb while retaining or enhancing the binding to FcgRIIaR andFcgRIIaH were produced by introducing alterations intoH240-Kn120/H240-H1068/L73-k0. In this examination, the present inventorstried to increase the binding activity to activating FcgRs: FcgRIIaR,FcgRIIaH, FcgRIIIaF, and FcgRIIIaV.

H240-Kn138 (SEQ ID NO: 116) resulting from introduction of L328W intoH240-Kn120; H240-Kn173 (SEQ ID NO: 117) resulting from introduction ofI332Q into H240-Kn120; H240-Kn178 (SEQ ID NO: 118) resulting fromintroduction of K334Y into H240-Kn120; H240-Kn166 (SEQ ID NO: 119)resulting from introduction of L328A into H240-Kn120; H240-Kn172 (SEQ IDNO: 120) resulting from introduction of I332M into H240-Kn120; andH240-Kn149 (SEQ ID NO: 121) resulting from introduction of L328W andK334L into H240-Kn120 were prepared according to the method described inReference Example 1. Furthermore, H240-H1147 (SEQ ID NO: 122) resultingfrom introduction of L328W into H240-H1068; H240-H1170 (SEQ ID NO: 123)resulting from introduction of L328A into H240-H1068; H240-H1174 (SEQ IDNO: 124) resulting from introduction of I332E into H240-H1068;H240-H1150 (SEQ ID NO: 125) resulting from introduction of I332T intoH240-H1068; H240-H1182 (SEQ ID NO: 126) resulting from introduction ofA231H into H240-H1068; and H240-H1177 (SEQ ID NO: 127) resulting fromintroduction of I332Q into H240-H1068 were prepared as the other Hchain. H240-Kn120/H240-H1170/L73-k0, H240-Kn120/H240-H1150/L73-k0,H240-Kn138/H240-H1068/L73-k0, H240-Kn120/H240-H1174/L73-k0,H240-Kn173/H240-H1068/L73-k0, H240-Kn178/H240-H1068/L73-k0,H240-Kn120/H240-H1182/L73-k0, H240-Kn138/H240-H1147/L73-k0,H240-Kn166/H240-H1170/L73-k0, H240-Kn172/H240-H1177/L73-k0, andH240-Kn149/H240-H1068/L73-k0 were prepared according to the methoddescribed in Reference Example 1 using L73-k0 (SEQ ID NO: 106) as the Lchain, H240-Kn138 (SEQ ID NO: 116), H240-Kn173 (SEQ ID NO: 117),H240-Kn178 (SEQ ID NO: 118), H240-Kn149 (SEQ ID NO: 121), H240-Kn166(SEQ ID NO: 119), and H240-Kn172 (SEQ ID NO: 120) as an H chain, andH240-H1170 (SEQ ID NO: 123), H240-H1150 (SEQ ID NO: 125), H240-H1174(SEQ ID NO: 124), H240-H1182 (SEQ ID NO: 126), H240-H1147 (SEQ ID NO:122), and H240-H1177 (SEQ ID NO: 127) as the other H chain. Thesevariants were assessed for the FcgR binding according to the methoddescribed in Reference Example 8. The results are shown in Table 60.

TABLE 60 ALTERA- ALTERA- TION TION INTRO- INTRO- FcgRIa FcgRIIaRFcgRIIaH FcgRIIb FcgRIIIaF FcgRIIIaV DUCED DUCED KD KD KD KD KD KD INTOINTO Sample (M) (M) (M) (M) (M) (M) H240-Kn120 H240-Hl068H240-Kn120/H240-Hl068/L73-k0 (SEQ ID 1.6E−10 2.0E−08 4.1E−08 1.5E−072.2E−09 3.6E−10 NO 99/SEQ ID NO 96/SEQ ID NO 106)H240-Kn120/H240-Hl170/L73-k0 (SEQ ID 1.9E−10 8.6E−09 1.8E−08 6.7E−083.9E−09 9.3E−10 L328A NO 99/SEQ ID NO 123/SEQ ID NO 106)H240-Kn120/H240-Hl150/L73-k0 (SEQ ID 3.9E−10 2.0E−08 3.5E−08 9.8E−083.0E−09 5.7E−10 I332T NO 99/SEQ ID NO 125/SEQ ID NO 106)H240-Kn138/H240-Hl068/L73-k0 (SEQ ID 1.6E−10 1.9E−08 2.5E−08 1.3E−073.7E−09 4.3E−10 L328W NO 116/SEQ ID NO 96/SEQ ID NO 106)H240-Kn120/H240-Hl174/L73-k0 (SEQ ID 1.4E−10 3.0E−08 5.5E−08 1.3E−075.1E−10 3.6E−11 I332E NO 99/SEQ ID NO 124/SEQ ID NO 106)H240-Kn173/H240-Hl068/L73-k0 (SEQ ID 1.2E−10 4.0E−08 5.7E−08 2.8E−071.8E−09 4.0E−10 I332Q NO 117/SEQ ID NO 96/SEQ ID NO 106)H240-Kn178/H240-Hl068/L73-k0 (SEQ ID 2.3E−10 2.6E−08 6.5E−08 1.1E−071.4E−09 1.9E−10 K334Y NO 118/SEQ ID NO 96/SEQ ID NO 106)H240-Kn120/H240-Hl182/L73-k0 (SEQ ID 1.8E−10 4.0E−08 7.7E−08 2.4E−073.1E−09 2.9E−10 A231H NO 99/SEQ ID NO 126/SEQ ID NO 106)H240-Kn138/H240-Hl147/L73-k0 (SEQ ID 3.1E−10 1.1E−08 1.3E−07 1.1E−072.2E−08 1.6E−08 L328W L328W NO 116/SEQ ID NO 122/SEQ ID NO 106)H240-Kn166/H240-Hl170/L73-k0 (SEQ ID 6.6E−11 3.1E−08 2.2E−08 2.0E−074.2E−09 1.0E−09 L328A L328A NO 119/SEQ ID NO 123/SEQ ID NO 106)H240-Kn172/H240-Hl177/L73-k0 (SEQ ID 2.5E−10 2.9E−08 5.7E−08 1.6E−072.0E−09 2.2E−10 I332M I332Q NO 120/SEQ ID NO 127/SEQ ID NO 106)H240-Kn149/H240-Hl068/L73-k0 (SEQ ID 3.8E−10 1.9E−08 2.6E−08 1.5E−071.5E−09 3.2E−10 L328W, K334L NO 121/SEQ ID NO 96/SEQ ID NO 106)

Furthermore, values obtained by dividing KD ofH240-Kn120/H240-H1068/L73-k0 for each of FcgRIa FcgRIIaR, FcgRIIaH,FcgRIIb, FcgRIIa F, and FcgRIIIa V by KD of each variant, the relativeKD determined when taking, as 1, KD of H240-Kn120/H240-H1068/L73-k0 foreach of FcgRIIaR, FcgRIIaH, and FcgRIIb are shown in Table 61.

TABLE 61 FcgRIa FcgRIIaR FcgRIIaH FcgRIIb FcgRIIIaF FcgRIIIaV RelativeRelative Relative Relative Relative Relative Sample KD KD KD KD KD KDH240-Kn120/H240-Hl068/L73-k0 (SEQ ID 1.0 1.0 1.0 1.0 1.0 1.0 NO 99/SEQID NO 96/SEQ ID NO 106) H240-Kn120/H240-Hl170/L73-k0 (SEQ ID 0.8 2.3 2.32.2 0.6 0.4 NO 99/SEQ ID NO 123/SEQ ID NO 106)H240-Kn120/H240-Hl150/L73-k0 (SEQ ID 0.4 1.0 1.2 1.5 0.7 0.6 NO 99/SEQID NO 125/SEQ ID NO 106) H240-Kn138/H240-Hl068/L73-k0 (SEQ ID 1.0 1.11.6 1.2 0.6 0.8 NO 116/SEQ ID NO 96/SEQ ID NO 106)H240-Kn120/H240-Hl174/L73-k0 (SEQ ID 1.1 0.7 0.7 1.2 4.3 10.0 NO 99/SEQID NO 124/SEQ ID NO 106) H240-Kn173/H240-Hl068/L73-k0 (SEQ ID 1.3 0.50.7 0.5 1.2 0.9 NO 117/SEQ ID NO 96/SEQ ID NO 106)H240-Kn178/H240-Hl068/L73-k0 (SEQ ID 0.7 0.8 0.6 1.4 1.6 1.9 NO 118/SEQID NO 96/SEQ ID NO 106) H240-Kn120/H240-Hl182/L73-k0 (SEQ ID 0.9 0.5 0.50.6 0.7 1.2 NO 99/SEQ ID NO 126/SEQ ID NO 106)H240-Kn138/H240-Hl147/L73-k0 (SEQ ID 0.5 1.8 0.3 1.4 0.1 0.0 NO 116/SEQID NO 122/SEQ ID NO 106) H240-Kn166/H240-Hl170/L73-k0 (SEQ ID 2.4 0.61.9 0.8 0.5 0.4 NO 119/SEQ ID NO 123/SEQ ID NO 106)H240-Kn172/H240-Hl177/L73-k0 (SEQ ID 0.6 0.7 0.7 0.9 1.1 1.6 NO 120/SEQID NO 127/SEQ ID NO 106) H240-Kn149/H240-Hl068/L73-k0 (SEQ ID 0.4 1.11.6 1.0 1.5 1.1 NO 121/SEQ ID NO 96/SEQ ID NO 106)

The variants shown in this table exhibit enhanced binding to at leastone of the FcgRs of FcgRIIaR, FcgRIIaH, FcgRIIIaF, and FcgRIIIaV ascompared to H240-Kn120/H240-H1068/L73-k0.

H240-Kn120/H240-H1170/L73-k0 resulting from introduction of L328A intoH240-H1068 that is an H chain of H240-Kn120/H240-H1068/L73-k0 hasFcgRIIaR- and FcgRIIaH-binding activities increased to 2.3 times ascompared to H240-Kn120/H240-H1068/L73-k0.

H240-Kn120/H240-H1150/L73-k0 resulting from introduction of I332T intoH240-H1068 that is an H chain of H240-Kn120/H240-H1068/L73-k0 hasFcgRIIaH-binding activity increased by 1.2 times that ofH240-Kn120/H240-H1068/L73-k0 while retaining the FcgRIIaR-bindingactivity.

H240-Kn138/H240-H1068/L73-k0 resulting from introduction of L328W intoH240-Kn120 that is an H chain of H240-Kn120/H240-H1068/L73-k0 hasFcgRIIaH-binding activity increased by 1.6 times that ofH240-Kn120/H240-H1068/L73-k0 while retaining the FcgRIIaR bindingactivity.

H240-Kn120/H240-H1174/L73-k0 resulting from introduction of I332E intoH240-H1068 that is an H chain of H240-Kn120/H240-H1068/L73-k0 hasFcgRIIIaF-binding activity increased by 4.3 times and FcgRIIIaV-bindingactivity increased to 10 times as compared toH240-Kn120/H240-H1068/L73-k0.

H240-Kn173/H240-H1068/L73-k0 resulting from introduction of I332Q intoH240-Kn120 that is an H chain of H240-Kn120/H240-H1068/L73-k0 hasFcgRIIIaF-binding activity increased by 1.2 times that ofH240-Kn120/H240-H1068/L73-k0 while retaining the FcgRIIIaV-bindingactivity.

H240-Kn178/H240-H1068/L73-k0 resulting from introduction of K334Y intoH240-Kn120 that is an H chain of H240-Kn120/H240-H1068/L73-k0 hasFcgRIIIaF-binding activity increased by 1.6 times and FcgRIIIaV-bindingactivity increased to 1.9 times as compared toH240-Kn120/H240-H1068/L73-k0.

H240-Kn120/H240-H1182/L73-k0 resulting from introduction of A231H intoH240-H1068 that is an H chain of H240-Kn120/H240-H1068/L73-k0 hasFcgRIIIaV-binding activity increased by 1.2 times as compared toH240-Kn120/H240-H1068/L73-k0.

H240-Kn138/H240-H1147/L73-k0 resulting from introduction of L328W intoboth H chains of H240-Kn120/H240-H1068/L73-k0 has FcgRIIaR-bindingactivity increased by 1.8 times as compared toH240-Kn120/H240-H1068/L73-k0.

H240-Kn166/H240-H1170/L73-k0 resulting from introduction of L328A intoboth H chains of H240-Kn120/H240-H1068/L73-k0 has FcgRIIaH-bindingactivity increased by 1.9 times as compared toH240-Kn120/H240-H1068/L73-k0.

H240-Kn172/H240-H1177/L73-k0 resulting from introduction of I332M intoH240-Kn120 that is an H chain of H240-Kn120/H240-H1068/L73-k0 andintroduction of I332Q into H240-H1068 that is the other H chain hasFcgRIIIaV-binding activity increased by 1.6 times that ofH240-Kn120/H240-H1068/L73-k0 while retaining the FcgRIIIaF-bindingactivity.

H240-Kn149/H240-H1068/L73-k0 resulting from introduction of L328W andK334L into H240-Kn120 that is an H chain of H240-Kn120/H240-H1068/L73-k0has FcgRIIaH-binding activity increased to 1.6 times that ofH240-Kn120/H240-H1068/L73-k0 while retaining the binding activities toFcgRIIaR and FcgRIIIaV.

The results described above suggest that the variants have strong ADCPor ADCC activity as compared to H240-Kn120/H240-H1068/L73-k0.

[Example 26] Preparation of Heterodimerized Antibody with IncreasedFcgRIIb-Binding Activity

In humans, FcγRIa (CD64A), FcγRIIa (CD32A), FcγRIIb (CD32B), FcγRIIIa(CD16A), and FcγRIIIb (CD16B) have been reported as isoforms of thehuman FcγR protein family, and their allotypes have also been reported(Immunol Lett, 82(1-2), 57-65, 2002). FcγRIa, FcγRIIa, and FcγRIIIa haveimmunoactivating functions, and are called activating FcγR, whileFcγRIIb has immunosuppressive functions, and is called inhibitory FcγR(Nat Rev Immunol, 10, 328-343, 2010).

FcγRIIb is the only FcγR expressed on B cells (Eur J Immunol 19,1379-1385, 1989). Interaction of the antibody Fc region with FcγRIIb hasbeen reported to suppress the primary immune response of B cells (J ExpMed 129, 1183-1201, 1969). Furthermore, it is reported that when FcγRIIbon B cells and a B cell receptor (BCR) are cross-linked via an immunecomplex in blood, B cell activation is suppressed, and antibodyproduction by B cells is suppressed (Immunol Lett 88, 157-161, 2003). Inthis immunosuppressive signal transduction mediated by BCR and FcγRIIb,the immunoreceptor tyrosine-based inhibitory motif (ITIM) contained inthe intracellular domain of FcγRIIb is necessary (Science, 256,1808-1812, 1992; Nature, 368, 70-73, 1994). When ITIM is phosphorylatedupon signaling, SH2-containing inositol polyphosphate 5-phosphatase(SHIP) is recruited, transduction of other activating FcγR signalcascades is inhibited, and inflammatory immune response is suppressed(Science, 290, 84-89, 2000). Furthermore, aggregation of FcgRIIb alonehas been reported to transiently suppress calcium influx due to BCRcross-linking and B cell proliferation in a BCR-independent mannerwithout inducing apoptosis of IgM-producing B cells (J Immunol, 181,5350-5359, 2008).

Furthermore, FcγRIIb is also expressed on dendritic cells, macrophages,activated neutrophils, mast cells, and basophils. FcγRIIb inhibits thefunctions of activating FcγR such as phagocytosis and release ofinflammatory cytokines in these cells, and suppresses inflammatoryimmune responses (Nat Rev Immunol, 10, 328-343, 2010).

The importance of immunosuppressive functions of FcγRIIb has beenelucidated so far through studies using FcγRIIb knockout mice. There arereports that in FcγRIIb knockout mice, humoral immunity is notappropriately regulated (J Immunol, 163, 618-622, 1999), sensitivitytowards collagen-induced arthritis (CIA) is increased (J Exp Med, 189,187-194, 1999), lupus-like symptoms are presented, and Goodpasture'ssyndrome-like symptoms are presented (J Exp Med, 191, 899-906, 2000).

Furthermore, regulatory inadequacy of FcγRIIb has been reported to berelated to human autoimmnue diseases. For example, the relationshipbetween genetic polymorphism in the transmembrane region and promoterregion of FcγRIIb, and the frequency of development of systemic lupuserythematosus (SLE) (Hum, Genet, 117, 220-227, 2005; J Biol Chem, 282,1738-1746, 2007; Arthritis Rheum, 54, 3908-3917, 2006; Nat Med, 11,1056-1058, 2005; J Immunol, 176, 5321-5328, 2006), and decrease ofFcγRIIb expression on the surface of B cells in SLE patients (J Exp Med,203, 2157-2164, 2006; J. Immunol. 178, 3272-3280, 2007) have beenreported.

From mouse models and clinical findings as such, FcγRIIb is consideredto play the role of controlling autoimmune diseases and inflammatorydiseases mainly through involvement with B cells, and it is a promisingtarget molecule for controlling autoimmune diseases and inflammatorydiseases.

IgG1, mainly used as a commercially available antibody pharmaceutical,is known to bind not only to FcγRIIb, but also strongly to activatingFcγR (Blood, 113, 3716-3725, 2009). It may be possible to developantibody pharmaceuticals having greater immunosuppressive propertiescompared with those of IgG1, by utilizing an Fc region with enhancedFcγRIIb binding, or improved FcγRIIb-binding activity selectivitycompared with activating FcγR. For example, it has been suggested thatthe use of an antibody having a variable region that binds to BCR and anFc with enhanced FcγRIIb binding may inhibit B cell activation (MolImmunol, 45, 3926-3933, 2008). It has been reported that crosslinkingFcγRIIb on B cells and IgE bound to a B-cell receptor suppressesdifferentiation of B cells into plasma cells, which as a result causessuppression of IgE production; and in human PBMC-transplanted mice,human IgG and IgM concentrations are maintained whereas the human IgEconcentration is decreased (Acad News, doi: 10.1016, jaci.2011.11.029).Besides IgE, it has been reported that when FcgRIIB and CD79b forming aB-cell receptor complex are cross-linked by an antibody, B cellproliferation is suppressed in vitro, and symptoms are alleviated in thecollagen arthritis model (Arthritis Rheum, 62, 1933-1943, 2010).

Besides B cells, it has been reported that crosslinking of FcεRI andFcgRIIb on mast cells using molecules, in which the Fc portion of an IgGwith enhanced FcgRIIb binding is fused to the Fc portion of IgE thatbinds to an IgE receptor FcεRI, causes FcgRIIb phosphorylation, therebysuppressing FcεRI-dependent calcium influx. This suggests thatinhibition of degranulation via FcgRIIb stimulation is possible byenhancing FcgRIIb binding (Immunol let, doi:10.1016/j.imlet.2012.01.008).

Accordingly, an antibody having an Fc with improved FcγRIIb-bindingactivity is suggested to be promising as a therapeutic agent forinflammatory diseases such as autoimmune diseases.

Furthermore, mutants with enhanced FcgRIIb binding have been suggestedto be promising therapeutic agents for cancer, as well as therapeuticagents for inflammatory diseases such as autoimmune diseases. So far,FcgRIIb has been found to play an important role in the agonisticactivity of agonist antibodies against the anti-TNF receptor family.Specifically, it has been suggested that interaction with FcgRIIb isrequired for the agonistic activity of antibodies against CD40, DR4,DR5, CD30, and CD137, which are included in the TNF receptor family(Science, 333, 1030-1034, 2011; Cancer Cell 19, 101-1113, 2011; J ClinInvest 2012, doi:10.1172/JCI61226; J Immunol 171, 562-, 2003; Blood,108, 705-, 2006; J Immunol 166, 4891, 2001). Non-patent Document(Science, 333, 1030-1034, 2011) shows that the use of antibodies withenhanced FcgRIIb binding enhances the anti-tumor effect of anti-CD40antibodies. Accordingly, antibodies with enhanced FcgRIIb are expectedto have an effect of enhancing agonistic activity of agonist antibodiesincluding antibodies against the anti-TNF receptor family.

Antibodies having an Fc with improved FcγRIIb-binding activity have beenreported (Mol Immunol, 45, 3926-3933, 2008). In this Document,FcγRIIb-binding activity was improved by adding alterations such asS267E/L328F, G236D/S267E, and S239D/S267E to an antibody Fc region. Inthe document, the antibody introduced with the S267E/L328F mutation moststrongly binds to FcγRIIb. Therefore, by further enhancing FcγRIIbbinding, it is expected that the above-described function mediated byFcγRIIb can be enhanced.

Furthermore, antibodies introducing S267E/L328E mutation maintains thesame level of binding to FcγRIa and FcγRIIa type H as that of anaturally-occurring IgG1. However, another report shows that thisalteration enhances the binding to type-R FcγRIIa several hundred timesto the same level of FcγRIIb binding, which means the FcγRIIb-bindingselectivity is not improved in comparison with type-R FcγRIIa (Eur JImmunol 23, 1098-1104, 1993).

Even if FcγRIIb binding had been enhanced compared with that of IgG1,only the effect of enhancing FcγRIIa binding and not the enhancement ofFcγRIIb binding is considered to have influence on cells such asplatelets which express FcγRIIa but do not express FcγRIIb (Nat RevImmunol, 10, 328-343, 2010). For example, the group of patients who wereadministered bevacizumab, an antibody against VEGF, is known to have anincreased risk for thromboembolism (J Natl Cancer Inst, 99, 1232-1239,2007). Furthermore, thromboembolism has been observed in a similarmanner in clinical development tests of antibodies against the CD40ligand, and the clinical study was discontinued (Arthritis Rheum, 48,719-727, 2003). In both cases of these antibodies, later studies usinganimal models and such have suggested that the administered antibodiesaggregate platelets via FcgRIIa binding on the platelets, and form bloodclots (J Thromb Haemost, 7, 171-181, 2008; J Immunol, 185, 1577-1583,2010). In systemic lupus erythematosus which is an autoimmune disease,platelets are activated via an FcγRIIa-dependent mechanism, and plateletactivation has been reported to correlate with the severity of symptoms(Sci Transl Med, vol 2, issue 47, 47-63, 2010). Even if FcgRIIb bindingis enhanced, administering an antibody with enhanced FcgRIIa binding tosuch patients who already have a high risk for developingthromboembolism will increase the risk for developing thromboembolism,thus is extremely dangerous.

Furthermore, antibodies with enhanced FcgRIIa binding have been reportedto enhance macrophage-mediated antibody dependent cellular phagocytosis(ADCP) (Mol Cancer Ther 7, 2517-2527, 2008). When antibody's antigensare phagocytized by macrophages, antibodies themselves are alsophagocytized at the same time. In that case, peptide fragments derivedfrom those antibodies are also presented as an antigen and theantigenicity may become higher, thereby increasing the risk ofproduction of antibodies against antibodies (anti-antibodies). Morespecifically, enhancing FcgRIIa binding will increase the risk ofproduction of antibodies against the antibodies, and this willremarkably decrease their value as pharmaceuticals.

More specifically, the value as pharmaceuticals will be considerablyreduced when FcgRIIa binding is enhanced, which leads to increased riskof thrombus formation via platelet aggregation, higher antigenicity, andincreased risk of anti-antibody production.

From such a viewpoint, the aforementioned Fc with enhanced FcgRIIbbinding shows remarkably enhanced type-R FcgRIIa binding compared withthat of a naturally-occurring IgG1. Therefore, its value as apharmaceutical for patients carrying type-R FcgRIIa is considerablyreduced. Types H and R of FcγRIIa are observed in Caucasians andAfrican-Americans with approximately the same frequency (J Clin Invest,97, 1348-1354, 1996; Arthritis Rheum, 41, 1181-1189, 1998). Therefore,when this Fc was used for treatment of autoimmune diseases, the numberof patients who can safely use it while enjoying its effects as apharmaceutical will be limited.

Furthermore, in dendritic cells deficient in FcgRIIb or dendritic cellsin which the interaction between FcgRIIb and the antibody Fc portion isinhibited by an anti-FcgRIIb antibody, dendritic cells have beenreported to mature spontaneously (J Clin Invest 115, 2914-2923, 2005;Proc Natl Acad Sci USA, 102, 2910-2915, 2005). This report suggests thatFcgRIIb is actively suppressing maturation of dendritic cells in asteady state where inflammation and such are not taking place. FcgRIIais expressed on the dendritic cell surface in addition to FcgRIIb;therefore, even if binding to inhibitory FcgRIIb is enhanced and ifbinding to activating FcgR such as FcgRIIa is also enhanced, maturationof dendritic cells may be promoted as a result. More specifically,improving not only the FcgRIIb-binding activity but also the ratio ofFcgRIIb-binding activity relative to FcgRIIa-binding activity isconsidered to be important in providing antibodies with animmunosuppressive action.

Therefore, when considering generation of pharmaceuticals that utilizethe FcgRIIb binding-mediated immunosuppressive action, there is a needfor an Fc that not only has enhanced FcgRIIb-binding activity, but alsohas binding to both FcgRIIa, types H and R allotypes, which ismaintained at a similar level or is weakened to a lower level than thatof a naturally-occurring IgG1.

Meanwhile, cases where amino acid alterations were introduced into theFc region to increase the FcγRIIb-binding selectivity have been reportedso far (Mol Immunol, 40, 585-593, 2003). However, all variants said tohave improved FcγRIIb selectivity as reported in this document showeddecreased FcγRIIb binding compared with that of a naturally-occurringIgG1. Therefore, it is considered to be difficult for these variants toactually induce an FcγRIIb-mediated immunosuppressive reaction morestrongly than IgG1.

Furthermore, since FcgRIIb plays an important role in the agonistantibodies mentioned above, enhancing their binding activity is expectedto enhance the agonistic activity. However, when FcgRIIa binding issimilarly enhanced, unintended activities such as ADCC activity and ADCPactivity will be exhibited, and this may cause side effects. Also fromsuch viewpoint, it is preferable to be able to selectively enhanceFcgRIIb-binding activity.

From these results, in producing antibody pharmaceuticals to be used fortreating autoimmune diseases and cancer utilizing FcγRIIb, it isimportant that compared with those of a naturally-occurring IgG, theactivities of binding to both FcγRIIa allotypes are maintained ordecreased, and FcγRIIb binding is enhanced. However, FcγRIIb shares 93%sequence identity in the extracellular region with that of FcγRIIa whichis one of the activating FcγRs, and they are very similar structurally.There are allotypes of FcγRIIa, H type and R type, in which the aminoacid at position 131 is His (type H) or Arg (type R), and yet each ofthem reacts differently with the antibodies (J Exp Med, 172, 19-25,1990). Therefore, to produce an Fc region that selectively binds toFcγRIIb, the most difficult problem may be conferring to the antibody Fcregion with the property of selectively improved FcγRIIb-bindingactivity, which involves distinguishing these homologous sequences, anddecreasing or not increasing the binding activity towards each allotypeof FcγRIIa, while increasing the binding activity towards FcγRIIb. Sofar, variants having sufficient FcgRIIb selectivity have not beenobtained. US 2009/0136485 reports variants with enhanced FcγRIIb-bindingactivity; however, the degree of enhancement is low, and there is ademand for development of variants having properties similar to thosedescribed above.

In this assessment, the present inventors investigated the preparationof variants whose binding activity has been increased in anFcgRIIb-selective manner using heterodimerized antibodies. Variableregion IL6R (SEQ ID NO: 128), which is the variable region of anantibody against human interleukin 6 receptor disclosed in WO2009/125825, was used as the variable region of antibody H chain.Furthermore, IL6R-G1d (SEQ ID NO: 129) containing G1d that lacks theC-terminal Gly and Lys of human IgG1 was produced, and then K439E wasintroduced into IL6R-G1d to construct IL6R-B3 (SEQ ID NO: 130).Meanwhile, E233D, G237D, P238D, H268D, P271G, and A330R were introducedinto IL6R-B3 to construct IL6R-BP208 (SEQ ID NO: 131). In addition,S267E and L328F were introduced into IL6R-B3 to construct IL6R-BP253(SEQ ID NO: 132) having an existing Fc with increased FcgRIIb-bindingactivity. The L chain of tocilizumab, IL6R-L (SEQ ID NO: 133), was usedas the common antibody L chain, and were used in combination with each Hchain according to the method described in Reference Example 1 toproduce homodimers IL6R-B3/IL6R-L, IL6R-BP208/IL6R-L, andIL6R-BP253/1L6R-L. These variants were assessed for the binding activityto FcgRIa, FcgRIIaR, FcgRIIaH, FcgRIIb, and FcgRIIIaV according to themethod described in Reference Example 8. The results are shown in Table62. The binding of FcgRIIaH and FcgRIIIaV to IL6R-BP208/IL6R-L and thebinding of FcgRIIIaV to IL6R-BP253/IL6R-L were concluded to be too weakto properly analyze by kinetic analysis. Thus, when the interactionbetween each of the altered antibodies and FcgR was weak, and correctanalysis was determined to be impossible by the above-mentioned kineticanalysis, the KD for such interactions were calculated (and thecalculated KD shown in Table 62) using the following 1:1 binding modelequation described in the Biacore T100 Software Handbook BR1006-48Edition AE.

The behavior of interacting molecules according to the 1:1 binding modelon Biacore can be described by Equation 1 shown below.R _(eq) =C·R _(max)/(KD+C)+RI  [Equation 1]R_(eq): a plot of steady state binding levels against analyteconcentrationC: concentrationRI: bulk refractive index contribution in the sampleR_(max): analyte binding capacity of the surface

When this equation is rearranged, KD can be expressed as Equation 2shown below.KD=C·R _(max)/(R _(eq) −RI)−C  [Equation 2]

KD can be calculated by substituting the values of R_(max), RI, and Cinto this equation. The values of RI and C can be determined from thesensorgram of the measurement results and measurement conditions.R_(max) was calculated according to the following method. As a target ofcomparison, for antibodies that had sufficiently strong interactions asevaluated simultaneously in the same round of measurement, the R_(max)value was obtained through global fitting using the 1:1 Langmuir bindingmodel, and then it was divided by the amount of the comparison antibodycaptured onto the sensor chip, and multiplied by the captured amount ofan altered antibody to be evaluated.

TABLE 62 FcgRIa FcgRIIaR FcgRIIaH FcgRIIb FcgRIIIaV sample KD (M) KD (M)KD (M) KD (M) KD (M) G1d 3.2E−10 1.0E−06 6.7E−07 2.6E−06 3.5E−07IL6R-B3/1L6R-L 4.2E−10 1.1E−06 7.7E−07 3.1E−06 3.3E−07 1L6R-BP253/5.0E−11 2.3E−09 8.6E−07 8.9E−09 7.1E−06 1L6R-L 1L6R-BP208/ 1.9E−088.5E−07 8.3E−06 3.2E−08 5.3E−05 1L6R-L

Values obtained by dividing KD of IL6R-B3/IL6R-L for each of FcgRIaFcgRIIaR, FcgRIIaH, and FcgRIIb by the corresponding KD of each modifiedvariant, the relative KD determined when taking, as 1, KD ofIL6R-B3/IL6R-L for FcgRIIaR, FcgRIIaH, or FcgRIIb are shown in Table 63.

TABLE 63 FcgRIIaR FcgRIIaH FcgRIIb Relative Relative Relative sample KDKD KD IL6R-B3/IL6R-L 1.0 1.0 1.0 IL6R-BP253/IL6R-L 480.0 0.9 349.2IL6R-BP208/IL6R-L 1.3 0.1 95.4

As shown in Table 63, IL6R-BP253/IL6R-L, which is an existing antibodywith increased FcgRIIb-binding activity, has FcgRIIb-binding activityincreased to about 350 times and FcgRIIaR-binding activity increased toabout 500 times as compared to human IgG1 antibody (IL6R-B3/IL6R-L)without introduction of the alteration. Meanwhile, the FcgRIIb-bindingactivity of IL6R-BP208/IL6R-L is about 100 times and thus inferior tothe existing antibody with increased FcgRIIb-binding activity; however,its FcgRIIaR-binding activity is 1.3 times and comparable to that of theIgG1 type. Thus, IL6R-BP208/IL6R-L is an antibody excellent inselectivity for FcgRIIb.

Next, the present inventors considered that, for the purpose ofimproving the FcgR2b-binding activity and the selectivity, it isnecessary to obtain information on the crystallographic structure of thecomplex between FcgRIIb extracellular region and Fc (BP208), which isthe Fc of IL6R-BP208/IL6R-L, and to more precisely assess amino acidmutations to be introduced. Thus, the complex between Fc (BP208) andFcgRIIb extracellular region was analyzed by X-ray crystallographyaccording to the experimental method described below.

[Expression and Purification of Fc (BP208)]

An Fc (BP208) was prepared as follows. First, Cys at position 220 (EUnumbering) of IL6R-BP208 was substituted with Ser. Then, geneticsequence of Fc (BP208) from Glu at position 236 (EU numbering) to its Cterminal was cloned by PCR. Using this cloned genetic sequence,production of expression vectors, and expression and purification of Fc(BP208) were carried out according to the method of Reference Example 1.Cys at position 220 (EU numbering) forms a disulfide bond with Cys ofthe L chain in general IgG1. The L chain is not co-expressed when Fcalone is prepared, and therefore, this residue was substituted with Serto avoid formation of unnecessary disulfide bonds.

[Expression and Purification of the FcgRIIb Extracellular Region]

This was prepared according to the method of Reference Example 8.

[Purification of the Fc (BP208)/FcgRIIb Extracellular Region Complex]

To 1.5 mg of the FcgRIIb extracellular region sample obtained forcrystallization, 0.15 mg of Endo Fl (Protein Science 1996, 5: 2617-2622)expressed and purified from Escherichia coli as a glutathioneS-transferase fusion protein was added. This was allowed to remain atroom temperature for three days in 0.1 M Bis-Tris buffer at pH 6.5, andthe N-linked oligosaccharide was cleaved, leaving N-acetylglucosaminedirectly bound to Asn. Next, this FcgRIIb extracellular region samplesubjected to carbohydrate cleavage treatment was concentrated byultrafiltration with 5000 MWCO, and purified by gel filtrationchromatography (Superdex200 10/300) using a column equilibrated in 20 mMHEPES at pH 7.5 containing 0.1 M NaCl. Furthermore, to the obtainedcarbohydrate-cleaved FcgRIIb extracellular region fraction, Fc(BP208)was added so that the molar ratio of the FcgRIIb extracellular regionwould be present in slight excess, and after concentration byultrafiltration with 10,000 MWCO, a sample of the Fc (BP208)/FcgRIIbextracellular region complex was obtained through purification by gelfiltration chromatography (Superdex200 10/300) using a columnequilibrated in 25 mM HEPES at pH 7.5 containing 0.1 M NaCl.

[Crystallization of the Fc (BP208)/FcgRIIb Extracellular Region Complex]

A sample of Fc (BP208)/FcgRIIb extracellular region complex wereconcentrated to about 10 mg/ml using 10000MWCO ultrafiltration filter,and crystallized using the hanging drop vapor diffusion method incombination with the seeding method. VDXm plate (Hampton Research) wasused for crystallization. Using a reservoir solution containing 0.1 MBis-Tris (pH 6.5), 19% PEG3350, and 0.2 M potassium phosphate dibasic,crystallization drops were prepared at a mixing ratio of reservoirsolution:crystallization sample=0.85 μl:0.85 μl. Crystals of the complexobtained under the same condition were crushed with Seed Bead (HamptonResearch) to prepare a seed crystal solution. 0.15 ul of a dilutedsolution produced from the seed crystal solution was added to thecrystallization drops, which were sealed in the wells containingreservoirs, and allowed to stand at 20° C., This successfully yieldedplate-like crystals.

[Measurement of X-Ray Diffraction Data from an Fc (BP208)/FcgRIIbExtracellular Region Complex Crystal]

One of the obtained single crystals of the Fc (BP208)/FcgRIIbextracellular region complex was soaked into a solution of 0.1 MBis-Tris pH 6.5, 24% PEG3350, 0.2 M potassium phosphate dibasic, ethleneglycol 20% (v/v). The crystal was fished out of the solution using a pinwith attached tiny nylon loop, and frozen in liquid nitrogen; and thenX-ray diffraction data was measured by BL32XUin Spring-8. During themeasurement, the crystal was constantly placed in a nitrogen stream at−178° C. to maintain in a frozen state, and a total of 300 X raydiffraction images were collected using MX-225HE CCD detector (RAYONIX)attached to a beam line with rotating the crystal 0.6° at a time.Determination of cell parameters, indexing of diffraction spots, anddiffraction data processing from the obtained diffraction images wereperformed using the Xia2 program (J. Appl. Cryst. 2010, 43: 186-190),XDS Package (Acta. Cryst. 2010, D66: 125-132) and Scala (Acta. Cryst.2006, D62: 72-82); and finally, diffraction intensity data up to 2.81 Åresolution was obtained. The crystal belongs to the space group C222₁,and has the following cell parameters; a=156.69 Å, b=260.17 Å, c=56.85Å, α=90°, β=90°, γ=90°.

[X Ray Crystallographic Analysis of the Fc (BP208)/FcgRIIb ExtracellularRegion Complex]

Crystal structure of the Fc (BP208)/FcγRIIb extracellular region complexwas determined by the molecular replacement method using the programPhaser ((J. Appl. Cryst. 2007, 40: 658-674). From the size of theobtained crystal lattice and the molecular weight of the Fc(BP208)/FcgRIIb extracellular region complex, the number of complexes inthe asymmetric unit was predicted to be one. From the structuralcoordinates of PDB code: 3SGJ which is the crystal structure of theFc(WT)/FcgRIIIa extracellular region complex, the amino acid residueportions of the A chain positions 239-340 and the B chain positions239-340 were taken out as separate coordinates, and they were usedrespectively as models for searching the Fc CH2 domains. The amino acidresidue portions of the A chain positions 341-444 and the B chainpositions 341-443 were taken out as a single set of coordinates from thesame structural coordinates of PDB code: 3SGJ; and this was used as amodel for searching the Fc CH3 domains. Finally, from the structuralcoordinates of PDB code: 2FCB which is a crystal structure of theFcgRIIb extracellular region, the amino acid residue portions of the Achain positions 6-178 was taken out and used as a model for searchingthe Fc (BP208). The present inventors tried to determine theorientations and positions of each search model of Fc CH3 domains,FcgRIIb extracellular region, and Fc CH2 domain in the crystal latticesusing rotation function and translation function, but failed todetermine the position of one of the CH2 domains. Then, with referenceto the crystal structure of the complex of Fc (WT)/FcgRIIIaextracellular region, the position of the last CH2 domain A wasdetermined from an electron density map that was calculated based on thephase determined from the remaining three parts. Thus, the presentinventors obtained an initial model for the crystal structure of the Fc(BP208)/FcgRIIb extracellular region complex. When rigid body refinementwhich moves the two Fc CH2 domains, the two Fc CH3 domains, and theFcgRIIb extracellular region was performed on the obtained initialmodel, the crystallographic reliability factor, R value became 42.6%,and the Free R value became 43.7% to diffraction intensity data from 25Å to 3.0 Å at this point. Furthermore, structural refinement using theprogram REFMACS (Acta Cryst. 2011, D67, 355-367), and revision of themodel to observe the electron density maps whose coefficient have 2Fo-Fcor Fo-Fc, which are calculated based on the experimentally determinedstructural factor Fo, the calculated structural factor Fc and thecalculated phase using the model, was carried out by the Coot program(Acta Cryst. 2010, D66: 486-501), and model refinement was carried outby repeating these steps. Finally, as a result of incorporation of watermolecules into the model based on the electron density maps which use2Fo-Fc or Fo-Fc as the coefficient, and the following refinement, thecrystallographic reliability factor, R values and the Free R value ofthe model containing 4794 non-hydrogen atoms became 24.4% and 27.9% to27259 diffraction intensity data from 25 Å to 2.81 Å resolution,respectively.

The three-dimensional structure of the complex of Fc (BP208)/FcgRIIbextracellular region was determined at 2.81 Å resolution by thestructure analysis. The structure obtained as a result of the analysisis shown in FIG. 49. FcgRIIb extracellular region was revealed to bebound and sandwiched between two Fc CH2 domains, which resembles thethree-dimensional structures of the previously analyzed complexesbetween Fc (WT), which is Fc of native IgG, and the respectiveextracellular regions of FcgRIIIa (Proc. Natl. Acad. Sci. USA, 2011,108, 12669-126674), FcgRIIIb (Nature, 2000, 400, 267-273; J. Biol. Chem.2011, 276, 16469-16477), and FcgRIIa.

However, a detailed observation of Fc (BP208) revealed an alteration inthe loop structure at 233-239 following the hinge region in CH2 domain Adue to an influence of the introduced mutations G237D and P238D ascompared to Fc (WT) bound to FcgRIIa (FIG. 50). The result showed thatthe main chain amide of G237 in Fc (BP208) formed a strong hydrogen bondwith the side chain of Tyr160 in FcgRIIb. In FcgRIIa, Phe is presentinstead of this Tyr160, and is incapable of forming such a hydrogenbond. This suggests that the above described hydrogen bond has importantcontribution to the acquisition of the selectivity, i.e., improvement ofthe FcgRIIb-binding activity and reduction of the FcgRIIa binding.

Based on the results of the structural analysis, the present inventorsprecisely assessed possible alterations to further increase theactivity, and found S239 as a candidate for the alteration introductionsite. As shown in FIG. 51, Ser239 of CH2 domain B is located at aposition toward which Lys117 of FcgRIIb protrudes in a structurallynatural fashion. However, since the electron density was not observedfor Lys117 of FcgRIIb by the analysis described above, it is thoughtthat Lys 117 does not take a definite structure and has only a limitedeffect on the interaction with Fc (BP208) in the current situation. WhenS239 of CH2 domain B is altered to negatively charged D or E, anelectrostatic interaction with positively charged Lys117 of FcgRIIb canbe expected, thereby resulting in improved FcgR11-binding activity.

On the other hand, an observation of the structure of S239 in CH2 domainA suggests that the side chain of this amino acid, by forming a hydrogenbond to the main chain of G236, stabilizes the loop structure atpositions 233 to 239, including D237 which forms a hydrogen bond withthe side chain of FcgRIIb Tyr160, following the hinge region (FIG. 52).The stabilization of the loop structure in the binding conformationsuppresses the entropy reduction upon binding, and results in anincrease in the binding free energy, leading to the improvement of thebinding activity. Meanwhile, when S239 of CH2 domain A is altered to Dor E, the loop structure becomes unstable due to loss of the hydrogenbond with the main chain of G236. In addition, electrostatic repulsionto D265 in close proximity may be caused, leading to furtherdestabilization of the loop structure. The energy for thedestabilization works to decrease the FcgRIIb-interaction energy,resulting in reduced binding activity. Specifically, in the case of ahomodimerized antibody both of whose H chains have been introduced withS239D or S239E, the binding activity-enhancing effect due to theelectrostatic interaction of CH2 domain B to Lys 117 of FcgRIIb iscounterbalanced by the binding activity-reducing effect due todestabilization of the loop structure in CH2 domain A, and thus may notresult in enhancement of the binding activity. On the other hand, in thecase of a heterodimerized antibody only one of whose H chains has beenintroduced with S239D or S239E, since the loop structure stabilizationby S239 of CH2 domain A is maintained, the binding activity was thoughtto increase corresponding to the newly formed electrostatic interactionwith Lys117 of FcgRIIb due to S239D or S239E introduced into CH2 domainB.

In order to assess the above-described hypothesis, the present inventorstried to produce antibodies with further increased FcgRIIb-bindingactivity by introducing mutation S239D or S239E into the Fc region ofone H chain alone using IL6R-BP208/IL6R-L as a template. IL6R-BP256 (SEQID NO: 134) resulting from introduction of S239D into IL6R-BP208, andIL6R-BP257 (SEQ ID NO: 135) resulting from introduction of S239E intoIL6R-BP208 were produced as an antibody H chain. Furthermore, IL6R-A5(SEQ ID NO: 136) resulting from introduction of mutations D356K andH435R into IL6R-G1d, and IL6R-AP002 (SEQ ID NO: 137) resulting fromintroduction of E233D, G237D, P238D, H268D, P271G, and A330R intoIL6R-A5 (SEQ ID NO: 136) were produced as the other H chain. IL6R-L (SEQID NO: 133) which is the L chain from tocilizumab was used as the commonantibody L chain, and the homodimerized antibodies IL6R-B3/IL6R-L,IL6R-BP208/IL6R-L, IL6R-BP253/IL6R-L, IL6R-BP256/IL6R-L, andIL6R-BP257/IL6R-L, and the heterodimerized antibodiesIL6R-AP002/IL6R-BP256/IL6R-L, and IL6R-AP002/IL6R-BP257/IL6R-L wereprepared together with the respective H chains according to the methoddescribed in Reference Example 1. The variants were assessed for thebinding activity to FcgR Ia, FcgR IIaR, FcgR IIaH, and FcgRIIb accordingto the method described in Reference Example 8. The results are shown inTable 64.

TABLE 64 FcgRIa FcgRIIaR FcgRIIaH FcgRIIb FcgRIIIaV sample KD (M) KD (M)KD (M) KD (M) KD (M) IL6R-B3/IL6R-L 4.2E−10 1.1E−06 7.7E−07 3.1E−063.3E−07 IL6R-BP253/IL6R-L 5.0E−11 2.3E−09 8.6E−07 8.9E−09 4.0E−07IL6R-BP208/IL6R-L 1.9E−08 8.5E−07 8.3E−06 3.2E−08 5.3E−05IL6R-BP256/IL6R-L 2.0E−09 7.3E−07 2.1E−05 8.7E−08 1.1E−05IL6R-BP257/IL6R-L 3.1E−10 1.3E−06 3.9E−05 4.3E−07 1.9E−05IL6R-AP002/IL6R-BP256/IL6R-L 3.6E−09 1.4E−07 4.2E−06 4.1E−09 1.7E−05IL6R-AP002/IL6R-BP257/IL6R-L 1.9E−09 1.3E−07 4.9E−06 4.7E−09 1.4E−05

The binding of FcgRIIaH to IL6R-BP208/IL6R-L, IL6R-BP256/IL6R-L, andIL6R-BP257/IL6R-L and the binding of FcgRIIIaV to IL6R-BP208/IL6R-L,IL6R-BP256/IL6R-L, IL6R-BP257/IL6R-L, IL6R-AP002/IL6R-BP256/IL6R-L, andIL6R-AP002/IL6R-BP257/IL6R-L were concluded to be too weak to properlyanalyze by kinetic analysis. Thus, when the interaction between each ofthe altered antibodies and FcgR was weak, and correct analysis wasdetermined to be impossible by the above-mentioned kinetic analysis, theKD for such interactions were calculated (and the calculated KD shown inTable 64) using the following 1:1 binding model equation described inthe Biacore T100 Software Handbook BR1006-48 Edition AE.

The behavior of interacting molecules according to the 1:1 binding modelon Biacore can be described by Equation 1 shown below.R _(eq) =C·R _(max)/(KD+C)+RI  [Equation 1]R_(eq): a plot of steady state binding levels against analyteconcentrationC: concentrationRI: bulk refractive index contribution in the sampleR_(max): analyte binding capacity of the surface

When this equation is rearranged, KD can be expressed as Equation 2shown below.KD=C·R _(max)/(R _(eq) −RI)−C  [Equation 2]

KD can be calculated by substituting the values of R_(max), RI, and Cinto this equation. The values of RI and C can be determined from thesensorgram of the measurement results and measurement conditions.R_(max) was calculated according to the following method. As a target ofcomparison, for antibodies that had sufficiently strong interactions asevaluated simultaneously in the same round of measurement, the R_(max)value was obtained through global fitting using the 1:1 Langmuir bindingmodel, and then it was divided by the amount of the comparison antibodycaptured onto the sensor chip, and multiplied by the captured amount ofan altered antibody to be evaluated.

Meanwhile, values obtained by dividing KD of IL6R-B3/IL6R-L for each ofFcgRIa FcgRIIaR, FcgRIIaH, and FcgRIIb by KD of each variant, therelative KD determined when taking, as 1, KD of IL6R-B3/IL6R-L for eachof FcgRIIaR, FcgRIIaH, and FcgRIIb, and values obtained by dividing KDof each variant for FcgRIIaR by KD for FcgRIIb are shown in Table 65.

TABLE 65 FcgRIIaR FcgRIIaH FcgRIIb Relative Relative Relative KD(IIaR)/sample KD KD KD KD(IIb) IL6R-B3/IL6R-L 1.0 1.0 1.0 0.3 IL6R-BP253/IL6R-L480.0 0.9 349.2 0.3 IL6R-BP208/IL6R-L 1.3 0.1 95.4 26.3IL6R-BP256/IL6R-L 1.5 0.0 35.7 8.4 IL6R-BP257/IL6R-L 0.9 0.0 7.2 3.0IL6R-AP002/ 7.7 0.2 751.8 34.3 IL6R-BP256/IL6R-L IL6R-AP002/ 8.3 0.2657.4 27.7 IL6R-BP257/IL6R-L

As shown in Table 65, IL6R-BP256/IL6R-L and IL6R-BP257/IL6R-L resultingfrom introduction of S239D or S239E into both H chains ofIL6R-BP208/IL6R-L exhibited reduced FcgRIIb-binding activity as well asreduced FcgRIIaR-binding activity as compared to the variantIL6R-BP208/IL6R-L before introduction. Meanwhile, the binding to FcgRIIbof IL6R-AP002/IL6R-BP256/IL6R-L and IL6R-AP002/IL6R-BP257/IL6R-Lresulting from introduction of S239D or S239E into one H chain ofIL6R-BP208/IL6R-L was increased by 752 times and 657 times,respectively, and thus their FcgRIIb-binding activities were higher thanthat achieved by the existing technique. Furthermore, theFcgRIIaR-binding activities were increased to 7.7 times and 8.3 times,respectively, while that of IL6R-BP208/IL6R-L was increased to 1.3times. In the table, KD(IIaR)/KD(IIb) represents a value obtained bydividing KD of each variant for FcgRIIaR by KD for FcgRIIb. When thevalue is larger, it means that the selectivity for FcgRIIb is higher.The value for IL6R-BP253/IL6R-L which is an existing antibody withincreased FcgRIIb-binding activity is 0.3, suggesting that theselectivity has not been improved as compared to IgG1 type, while thevalue for IL6R-BP208/IL6R-L is 26.3, implying that it has high FcgRIIbselectivity. In this experiment, IL6R-AP002/IL6R-BP256/IL6R-L andIL6R-AP002/IL6R-BP257/IL6R-L, resulting from introduction of S239D orS239E into one H chain of IL6R-BP208/IL6R-L, showed KD(IIaR)/KD(IIb)values of 34.3 and 27.7, respectively, and were improved more thanIL6R-BP208/IL6R-L.

The result of comprehensive assessment of variants using IgG1 type(GpH7-B3/GpL16-k0) as a template, which was carried out as described inExample 4, shows that S239D and S239E have effects as shown in Table 66.In the table, Ho/Con_2aR and Ho/Con_2b represent the FcgRIIaR- andFcgRIIb-binding activity levels when taking as 100 those of the controlhomodimerized antibody. Meanwhile, He/Con_2aR and He/Con_2b representthe FcgRIIaR- and FcgRIIb-binding levels when taking as 100 those of thecontrol heterodimerized antibody.

TABLE 66 ALTERA- TION Ho/Con_2aR Ho/Con_2b He/Con_2aR He/Con_2b S239D137 223 129 209 S239E 117 174 127 183

Table 66 shows that alterations S239D and S239E introduced in thisexperiment, when introduced into native IgG1 regardless of either orboth chains, enhanced the binding to FcRIIaR and FcgRIIb. However, thealterations, when introduced into both H chains of IL6R-BP208/IL6R-L,reduced the binding activities to FcgRIIaR and FcgRIIb, and only whenintroduced into one H chain, increased the binding activities toFcgRIIaR and FcgRIIb. The result demonstrates that the effect of thealterations is different from that obtained using the IgG1 type as atemplate. Thus, these alterations were demonstrated to achieve theabove-described effect only when introduced into IL6R-BP208/IL6R-L.

[Example 27] Design of the Amino Acid Sequence of Constant Region toImprove the Capacity to Separate and Purify Homodimers and Heterodimers

[Selection of Residue Substitution Site]

Co-expression of two types of H chains (referred to as A chain and Bchain) in manufacturing heterodimerized antibodies results in formationof: homodimerized antibody resulting from dimerization of H chains bothof which are A chains, homodimerized antibody resulting fromdimerization of H chains both of which are B chains, and heterodimerizedantibody resulting from dimerization of H chains one of which is A chainand the other is B chain. A known method for efficiently separating andpurifying heterodimerized antibodies of interest is to control theisoelectric point of an antibody and the capacity of retention andseparation in an ion-exchange column by substituting amino acid residuesin each variable region (WO 2007/114325). However, since each antibodyhas a different sequence in its variable regions, particularly in theCDR region, the method described above has limited versatility. Then, asa more versatile method for substituting residues in antibodies topurify heterodimerized antibodies, the present inventors investigated amethod for controlling the isoelectric point and the capacity ofretention and separation in an ion-exchange column by substitutingresidues only in the constant region of an antibody.

In general, the separation in an ion-exchange column is thought todepend on electric charges on the surface of molecules, and in manycases the separation condition is examined by considering theisoelectric point of a target molecule. Thus, in this Example, theseparation in an ion-exchange column was also expected to be improved bysubstituting amino acid residues that constitute the antibody constantregion in a way that causes an electric point difference betweenhomodimerized antibodies and heterodimerized antibodies to be separated.

Meanwhile, not only a pure ion-exchange mode but also hydrophobicinteraction has been suggested to be involved in the separation byion-exchange chromatography (Peng Liu et al., J Chromatogr A. 2009 Oct.30; 1216(44): 7497-504). For this reason, in the separation andpurification based on the method described above, it is possible to usehydrophobic chromatography in addition to ion-exchange chromatography.

Residue substitution methods for altering the isoelectric point includemethods for substituting a neutral residue with a basic or acidicresidue and methods for substituting a basic or acidic residue with aneutral residue. More effective approaches include methods forsubstituting a positively charged residue with a negatively chargedresidue and methods for substituting a negatively charged residue with apositively charged residue.

In the methods described above, every part of an antibody sequence canbe a candidate for the substitution site of a residue that results in anisoelectric point change. However, random substitution that gives riseto a non-native sequence can increase the risk of immunogenicity, and isnot an appropriate method from the viewpoint of pharmaceutical use.

Residues can be substituted so as to minimize the number of T-cellepitopes involved in immunogenicity, in order to minimize the increasein the risk of immunogenicity. One possible means includes the use ofIgG subclass sequences. Human IgG subclasses include IgG1, IgG2, IgG3,and IgG4. By substituting portions of an antibody sequence withsequences of different subclasses according to a method disclosed in WO2007/114325, the isoelectric point can be altered while suppressing theincrease in the number of T-cell epitopes.

Alternative methods available include in silico T-cell epitopeprediction tools such as Epibase.

Epibase Light (Lonza) is an in silico tool to calculate the bindingcapacity between 9-mer peptide and major DRB1 allele using FASTERalgorism (Expert Opin Biol Ther. 2007 March; 7(3): 405-18). This toolenables identification of T-cell epitopes that strongly or moderatelybind to MHC class II.

The calculation reflects the abundance ratio of DRB1 allotypes. For thispurpose, it is possible to use the following abundance ratio inCaucasian populations.

DRB1*1501(24.5%), DRB1*0301(23.7%), DRB1*0701(23.3%), DRB1*0101(15.0%),DRB1*1101(11.6%), DRB1*1302(8.2%), DRB1*1401/1454(4.9%),DRB1*0901(2.3%), DRB1*1502(0.5%), DRB1*1202(0.1%)

All epitopes in each modified antibody sequence that exhibit strong ormoderate binding are identified by FASTER algorism, and then criticalepitopes are displayed after excluding human germline sequences andsequence of the junction of variable region and constant region. Thenumber of T-cell epitopes increased by substitution of any amino acidresidue for each residue in any sequence is calculated by using therandomization function of this tool. This enables selection of sties ofresidue substitution that results in an isoelectric point change withoutincreasing the number of T-cell epitopes.

H240-AK072 (SEQ ID NO: 104) and H240-BH076 (SEQ ID NO: 105) wereanalyzed by Epibase. Table 67 shows as to H240-AK072 the number ofT-cell epitopes that can be changed by substitution of any residue,while Table 68 shows the number for H240-BH076. Based on the results,one can select residue substitution that results in an isoelectric pointchange without increasing T-cell epitopes.

[Table 67-1]

TABLE 67 EU AK072 No. sequence A C D E F G H I K L M N P Q R S T V W Y118 A 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 119 S 0 0 0 0 1 0 0 1 0 11 0 0 0 0 0 0 1 1 1 120 T 0 0 0 0 1 0 0 1 0 1 1 0 0 0 0 0 0 1 1 1 121 K0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 122 G 0 0 0 0 1 0 0 0 0 0 0 0 00 0 0 0 0 0 1 123 P 1 0 0 0 2 0 1 1 1 2 1 0 0 0 2 1 0 1 2 2 124 S 0 0 00 2 0 0 2 2 2 2 1 0 1 2 0 0 1 1 1 125 V 0 0 0 0 1 0 0 1 0 1 1 0 0 0 0 00 0 1 1 126 F 0 1 0 1 0 0 1 2 2 2 2 2 0 1 2 1 1 2 3 2 127 P 4 3 3 3 4 33 4 2 4 4 3 0 3 2 3 3 4 4 4 128 L 1 1 2 2 3 1 2 3 4 0 3 2 1 2 3 2 1 3 33 129 A 0 3 3 3 4 3 3 3 3 3 3 3 2 4 3 3 3 3 4 3 130 P 3 3 3 3 4 3 3 4 34 4 3 0 3 4 3 3 3 5 4 131 S 3 3 3 3 4 3 2 4 3 4 4 3 3 3 3 0 3 4 3 4 132S 3 3 3 3 4 3 3 4 3 4 4 3 3 3 3 0 3 4 4 4 133 K 3 2 3 3 3 2 3 3 0 3 3 33 3 3 3 3 3 3 3 134 S 3 3 3 3 4 3 3 4 3 4 5 3 3 3 3 0 3 4 4 4 135 T 2 22 1 3 2 2 4 2 4 4 2 2 2 3 2 0 2 3 3 136 S 1 1 1 1 2 1 1 2 1 2 3 1 1 1 10 1 1 2 2 137 G 1 1 1 1 2 0 1 2 1 3 2 1 1 1 1 1 1 2 2 2 138 G 0 1 0 0 10 0 1 1 2 1 0 0 0 1 0 0 1 1 1 139 T 1 1 0 0 3 0 1 3 1 3 3 0 0 1 1 0 0 31 3 140 A 0 0 0 0 1 0 1 1 1 1 1 0 0 0 1 0 0 1 1 1 141 A 0 0 0 0 2 0 0 20 1 1 0 0 0 0 0 0 0 2 2 142 L 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1143 G 2 0 1 1 2 0 1 2 1 2 2 1 1 2 2 1 1 2 2 2 144 C 2 0 0 1 4 2 3 5 2 55 2 2 3 3 2 1 4 4 4 145 L 0 0 1 1 0 0 1 0 1 0 0 0 0 1 1 1 0 0 1 0 146 V0 0 0 0 2 0 1 0 1 2 1 1 1 0 1 1 1 0 1 1 147 K 0 0 0 0 3 0 1 3 0 3 3 1 11 2 1 1 3 1 3 148 D 2 2 0 2 4 2 3 4 4 4 4 2 2 3 3 3 3 4 4 4 149 Y 1 0 10 1 0 2 2 1 3 3 2 1 0 3 2 1 2 2 0 150 F 2 1 2 2 0 1 3 2 3 2 1 2 2 3 4 43 2 1 2 151 P 3 2 2 2 3 2 3 4 4 5 5 3 0 4 3 3 2 2 3 3 152 E 4 2 1 0 4 24 3 3 3 3 3 2 3 4 5 2 3 4 4 153 P 3 2 3 3 3 1 3 5 2 5 5 1 0 2 4 3 2 3 34 154 V 2 1 1 1 2 1 2 2 2 2 2 1 1 2 2 2 1 0 1 2 155 T 3 2 2 2 4 2 3 4 34 4 2 2 2 3 3 0 4 4 4 156 V 2 2 1 2 3 2 2 3 2 3 3 2 2 2 3 2 2 0 3 3 157S 3 3 2 2 5 3 3 4 2 5 5 2 0 2 3 0 3 5 4 5 158 W 3 3 3 2 3 3 3 3 4 3 3 32 3 4 4 4 4 0 3 159 N 2 2 1 1 2 2 2 2 3 2 2 0 1 3 2 2 2 3 2 2 160 S 4 32 2 4 4 4 4 4 4 4 3 3 3 3 0 4 4 4 4 161 G 4 3 2 2 5 0 5 5 4 5 5 4 3 4 43 3 5 4 5 162 A 0 4 1 1 5 3 2 6 5 5 6 4 4 3 5 4 4 6 5 5 163 L 3 1 1 1 23 2 3 3 0 4 2 3 2 3 3 3 3 1 2 164 T 3 3 1 2 4 3 3 5 3 5 5 3 3 3 3 3 0 45 4 165 S 3 2 1 1 2 1 3 4 3 3 3 2 3 2 3 0 2 3 2 3 166 G 4 2 1 2 4 0 5 53 5 4 4 2 4 6 5 3 5 4 4 167 V 3 3 4 0 4 3 3 3 4 5 5 4 3 4 4 4 3 0 4 4168 H 2 1 1 1 3 2 0 3 3 3 3 2 1 1 3 1 2 2 2 3 169 T 3 3 2 1 4 3 4 5 3 44 4 2 4 5 3 0 4 4 4 170 F 3 1 1 1 0 1 1 3 3 2 3 3 2 3 2 2 2 3 1 3 171 P6 4 3 3 6 3 6 6 7 6 6 5 0 6 6 4 4 6 5 5 172 A 0 5 0 0 6 3 6 6 5 6 6 4 45 7 6 3 6 6 6 173 V 3 2 2 2 7 2 5 6 2 6 6 3 2 5 3 4 3 0 6 5 174 L 4 5 34 6 4 4 6 5 0 6 3 2 2 5 4 4 6 6 6 175 Q 5 4 5 4 5 5 7 6 7 6 6 5 4 0 7 66 5 6 6 176 S 7 6 3 4 6 6 5 6 6 6 6 5 5 5 6 0 6 6 6 0 177 S 6 3 3 4 5 45 6 5 5 5 5 3 3 5 0 5 6 6 5 178 C 8 7 5 4 8 0 8 8 8 8 8 6 7 7 9 7 7 8 88 179 L 4 6 4 2 7 5 6 7 6 0 7 6 5 7 6 6 5 7 7 7 180 Y 5 4 3 3 6 2 6 6 66 6 3 4 4 6 5 4 6 7 0 181 S 6 6 5 3 7 5 6 7 8 7 7 7 5 7 8 0 7 7 8 8 182L 6 6 3 4 7 5 6 7 5 0 7 6 5 6 8 7 5 7 6 7 183 S 6 5 4 3 6 6 5 6 7 6 6 56 7 7 0 6 6 6 6 184 S 6 5 4 4 7 5 7 8 7 7 7 5 4 6 7 0 6 6 6 6 185 V 4 54 3 6 4 5 6 5 6 6 6 3 5 5 5 5 0 5 6 186 V 4 4 4 5 5 4 5 5 5 5 5 4 4 4 55 4 0 5 6 187 T 6 5 6 5 8 5 6 8 6 8 8 5 5 7 6 5 0 8 7 8 188 V 4 3 4 4 44 4 4 4 4 4 3 3 3 5 4 4 0 4 4 189 P 6 4 4 4 5 4 5 7 6 7 7 4 0 5 6 4 3 56 7 190 S 2 3 2 2 3 2 2 3 2 3 3 2 2 3 2 0 2 3 2 3 191 S 2 2 2 2 3 2 2 33 4 3 2 2 2 4 0 2 3 3 2 192 S 2 1 1 1 3 1 3 3 2 3 3 2 1 2 3 0 1 3 2 3193 L 1 1 2 1 2 1 2 2 2 0 1 3 1 2 2 1 2 1 2 2 194 G 3 2 1 2 6 0 3 6 4 66 3 2 3 4 2 1 5 5 6 195 T 1 0 0 0 4 0 3 4 2 4 5 1 0 3 2 0 0 3 2 4 196 Q2 1 1 0 3 2 1 3 2 4 3 2 1 0 4 2 1 3 3 3 197 T 1 1 1 1 2 1 1 5 1 3 3 2 01 2 1 0 3 4 3 198 Y 2 1 0 0 2 2 1 3 2 3 4 2 2 3 2 1 2 3 2 0 199 I 2 1 12 3 2 2 0 2 3 3 2 1 2 2 3 0 3 3 3 200 C 5 0 2 3 6 4 6 7 6 6 6 5 6 6 6 55 7 4 6 201 N 5 3 3 3 5 4 5 4 4 5 5 0 1 4 5 4 4 3 4 5 202 V 4 3 2 2 5 36 5 5 6 5 5 2 6 5 4 3 0 4 5 203 N 4 3 3 2 5 2 5 6 5 5 5 0 3 3 6 5 5 5 45 204 H 5 3 4 4 5 5 0 6 5 6 6 5 3 6 4 5 3 6 4 5 205 K 4 4 2 4 5 4 4 6 06 5 5 2 4 5 4 4 5 5 5 206 P 5 3 2 2 5 4 3 5 5 6 5 6 0 5 6 5 5 5 4 5 207S 4 3 3 2 4 2 4 5 3 4 4 3 4 4 5 0 4 4 3 4 208 N 3 2 2 2 4 3 2 4 2 5 4 03 3 3 2 2 4 5 4 209 T 2 2 1 1 3 2 2 3 3 3 3 3 2 2 3 2 0 3 2 3 210 K 2 11 1 1 1 1 1 0 2 1 1 1 1 1 1 1 1 2 1 211 V 2 1 1 1 1 1 2 2 2 2 1 2 2 2 21 1 1 2 1 212 D 2 1 0 0 4 1 2 3 1 3 3 1 1 2 3 1 1 3 2 3 213 K 0 0 0 0 20 2 2 0 1 1 1 0 1 1 0 1 1 2 2 214 K 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 10 0 215 V 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 216 E 1 1 0 0 2 1 1 32 3 2 1 1 2 2 1 2 3 1 2 217 P 0 0 0 0 2 0 1 2 1 2 2 0 0 1 1 0 0 2 2 2218 K 0 0 0 0 2 0 0 2 0 2 2 0 0 1 0 1 0 2 2 2 219 S 1 0 0 0 0 0 0 1 0 10 0 0 1 0 0 0 0 0 0 220 C 0 0 0 0 0 0 1 1 2 1 1 1 1 1 3 2 1 0 1 1 221 D2 0 0 0 3 2 1 1 1 2 3 2 0 1 2 1 0 1 3 3 222 K 0 0 0 0 0 0 1 1 0 0 0 1 01 0 0 0 0 0 0 223 T 0 0 0 0 2 0 0 0 0 0 0 0 0 1 1 1 0 0 1 1 224 H 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 225 T 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 00 0 1 1 226 C 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 227 P 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 0 228 P 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 10 229 C 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 230 P 0 0 0 0 0 0 0 1 01 1 0 0 0 0 0 0 1 0 0 231 A 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 232P 0 0 −1 −1 0 0 −1 0 0 0 0 0 0 0 0 0 0 0 0 0 233 E 1 1 0 0 1 1 1 1 1 1 11 1 1 1 1 1 1 1 1 234 Y −1 −1 −2 −2 0 −1 −1 1 −1 0 0 −1 −1 0 0 −1 −1 0 00 235 Y −1 −1 −1 −1 0 −1 −1 −1 −1 1 0 −1 −1 −1 −1 −1 −1 −1 1 0 236 W −1−1 −1 −1 −1 −1 −1 −1 0 −1 −1 −1 −1 −1 0 −1 −1 −1 0 0 237 C 0 0 −1 −1 1 00 2 0 1 1 0 −1 0 3 0 −1 0 2 2 238 P 1 0 0 0 2 0 1 3 2 2 3 2 0 0 2 1 1 13 3 239 S −1 0 −1 −1 1 0 −1 1 1 1 1 −1 0 1 2 0 1 1 2 1 240 V −1 −1 −1 −11 −1 0 1 1 2 1 0 −1 −1 1 0 0 0 0 2 241 F 1 −1 −1 −1 0 −1 2 3 2 4 3 0 0−1 2 0 0 4 2 4 242 L 1 1 1 0 1 1 1 2 1 0 2 1 1 2 2 1 1 1 1 1 243 F 3 2 11 0 1 3 3 3 3 3 2 2 3 3 4 3 3 1 3 244 P 4 2 3 4 5 4 4 5 4 5 5 4 0 4 5 43 5 5 5 245 P 5 2 1 1 5 3 5 5 3 6 5 3 0 3 3 4 2 5 5 5 246 K 2 0 2 1 2 12 3 0 4 3 1 2 1 3 2 2 3 3 3 247 P 2 2 1 1 3 2 2 3 2 4 4 2 0 2 4 2 2 2 23 248 K 3 1 2 1 4 2 4 4 0 4 4 1 1 3 4 3 2 4 4 4 249 D 4 4 0 3 5 4 4 5 65 5 4 3 4 6 4 4 5 6 6 250 T 2 2 2 2 2 2 2 2 3 3 3 2 2 2 3 2 0 2 3 2 251L 2 2 1 1 3 1 2 2 2 0 3 3 1 1 3 3 3 2 2 3 252 M 3 3 1 2 3 3 3 3 3 3 0 33 3 4 3 3 3 3 4 253 I 4 3 3 2 3 2 3 0 4 3 3 4 3 3 4 4 4 3 3 3 254 S 4 33 3 4 3 3 4 3 4 4 4 3 4 3 0 4 4 4 4 255 R 3 2 2 2 4 2 4 5 3 4 4 3 3 3 04 3 4 3 3 256 T 3 3 3 3 5 3 3 5 3 5 5 3 3 4 3 4 0 5 5 5 257 P 4 3 3 2 54 4 5 3 5 5 4 0 5 3 4 4 5 5 5 258 E 4 3 3 0 4 3 3 4 5 4 5 4 4 3 4 4 4 54 4 259 V 2 2 2 2 3 2 2 3 2 3 3 2 2 2 2 2 2 0 3 3 260 T 2 1 2 2 3 2 2 33 2 3 2 1 2 3 2 0 2 3 2 261 C 2 0 0 0 3 0 1 4 1 3 3 1 0 0 1 1 1 2 2 3262 V −1 −1 −1 −1 1 −1 −1 0 0 0 0 −1 −1 0 0 0 −1 0 0 1 263 V 0 −1 −1 −1−1 −1 −1 0 −1 1 0 0 −1 −1 −1 −1 −1 0 0 0 264 V 0 −1 −1 −1 −1 −1 −1 0 0−1 0 0 0 0 1 0 0 0 −1 0 265 D 0 1 0 0 2 0 1 1 −1 1 1 1 0 1 −1 1 1 1 1 2266 V −1 0 −1 −1 0 −1 1 0 −1 1 0 −1 0 0 0 0 −1 0 −1 0 267 S −1 −1 0 0 −1−1 0 0 0 −1 −1 0 0 0 0 0 1 −1 −1 −1 268 D 0 −1 0 −1 1 1 −1 2 0 2 2 0 2 01 1 0 2 1 1 269 E 1 1 1 0 2 1 1 2 1 2 2 2 1 1 2 1 2 2 2 2 270 E 0 0 0 01 0 1 2 1 2 2 0 0 0 1 0 0 2 1 1 271 P 1 0 0 1 2 0 1 2 1 2 2 1 0 1 1 1 12 2 1 272 E 0 0 0 0 1 0 0 1 0 1 1 0 0 0 1 0 0 1 1 1 273 V 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 0 1 0 274 K 0 0 0 0 1 0 0 1 0 1 1 0 0 0 0 0 0 0 1 1275 F 0 0 0 0 0 0 0 1 1 1 1 0 0 0 0 0 0 1 1 1 276 N 1 1 1 1 3 1 2 3 1 33 0 1 2 2 2 2 3 3 3 277 W 1 1 0 0 2 1 1 2 2 2 2 1 0 1 2 2 2 2 0 2 278 Y2 1 1 0 2 1 2 2 3 3 2 2 2 3 3 3 3 3 2 0 279 V 2 2 1 1 2 2 2 2 2 2 2 2 23 2 2 2 0 2 2 280 D 4 3 0 2 6 3 4 5 6 5 5 3 2 5 6 4 4 5 5 7 281 G 3 3 22 3 0 3 4 3 5 5 3 2 3 4 3 2 3 3 3 282 V 3 2 1 2 2 2 3 2 3 2 0 2 3 2 4 32 0 2 2 283 E 4 3 2 0 6 4 3 6 5 7 6 4 4 4 7 4 4 7 5 6 284 V 3 2 1 1 3 22 3 3 4 3 4 2 2 2 2 2 0 3 3 285 H 4 3 2 2 4 3 0 4 3 4 4 3 2 4 3 3 2 4 34 286 N 3 3 2 2 4 3 3 4 3 5 4 0 3 3 4 3 3 3 4 4 287 A 0 2 2 1 2 2 2 2 32 2 2 1 2 3 2 1 2 3 3 288 K 1 0 0 0 2 1 3 3 0 3 3 3 2 1 2 2 2 3 2 2 289T 2 1 2 2 3 2 4 3 4 3 3 3 1 3 4 2 0 3 2 3 290 K 1 1 1 0 3 1 2 4 0 4 3 22 2 2 1 3 3 3 4 291 P 1 2 1 2 5 1 3 2 2 0 3 1 0 2 3 2 2 3 1 3 292 R 0 10 0 2 0 1 2 1 2 2 0 0 0 0 1 0 2 0 2 293 E 1 0 1 0 3 1 2 3 2 3 2 1 1 2 22 3 2 3 3 294 E 2 2 1 0 3 1 3 4 3 4 3 1 3 2 2 3 2 4 2 3 295 Q −1 −1 −1−1 1 −1 −1 1 −1 1 1 0 −1 0 −1 −1 −1 1 0 1 296 Y −2 −2 −2 −2 0 −2 0 0 0−1 −1 −2 −2 −2 0 −1 −2 −1 0 0 297 N 0 0 −1 −1 1 −1 2 0 1 1 1 0 −1 0 1 01 0 1 1 298 A 0 −1 −1 −1 2 −1 −1 2 2 2 2 0 −1 0 2 −2 −1 2 1 2 299 T 1 10 0 1 1 0 2 1 2 2 0 1 0 2 0 0 2 1 2 300 Y 2 2 −1 0 0 1 2 1 1 2 2 2 0 1 43 3 1 1 0 301 R 2 2 −1 0 3 2 2 3 1 4 4 1 1 2 0 1 1 3 3 4 302 V 4 3 2 2 33 3 3 3 3 3 4 2 3 4 4 3 0 3 3 303 V 3 3 3 2 4 3 3 5 5 5 5 4 3 3 3 4 4 05 5 304 S 4 4 3 3 5 4 5 6 5 5 5 4 4 7 6 0 4 7 5 5 305 V 4 4 3 4 5 4 4 54 5 6 4 4 4 4 4 4 0 5 5 306 L 8 6 3 3 7 6 6 7 7 0 7 6 6 6 7 6 6 7 6 7307 T 7 6 5 5 8 6 7 7 6 8 8 6 7 6 6 6 0 7 9 7 308 V 6 6 5 5 7 5 7 7 5 88 6 5 6 5 6 6 0 6 7 309 L 5 4 4 4 6 5 4 7 5 0 6 6 6 5 6 7 6 0 5 6 310 H5 3 3 2 6 4 0 6 5 6 5 4 5 7 6 6 5 5 5 6 311 Q 4 4 3 4 5 4 4 5 4 5 5 4 40 6 4 4 4 5 5 312 D 4 5 0 2 6 4 5 7 5 8 6 5 4 4 6 5 5 6 7 6 313 W 3 1 11 3 3 4 3 2 3 3 2 2 1 3 3 3 3 0 3 314 L 2 2 2 2 3 2 3 3 4 0 3 3 3 2 4 24 3 3 3 315 N 2 2 1 1 2 2 3 4 2 4 3 0 2 2 3 3 2 3 2 2 316 G 1 1 0 0 3 01 2 0 3 2 2 0 2 2 0 0 2 2 4 317 K 1 1 0 0 2 1 2 2 0 2 1 1 1 1 4 2 1 1 22 318 E 2 1 2 0 3 2 2 4 2 3 4 2 2 2 3 3 4 3 4 4 319 Y 2 1 1 1 2 1 1 2 32 2 1 1 1 3 1 1 2 2 0 320 K 3 2 1 1 4 3 2 4 0 4 4 3 2 3 3 2 2 4 3 4 321C 3 0 2 3 4 3 4 3 4 3 3 2 2 3 5 4 3 3 3 4 322 K 3 1 1 1 5 2 4 5 0 5 5 23 2 4 2 4 5 4 5 323 V 3 2 2 2 3 2 2 3 3 3 3 5 3 4 3 3 2 0 3 3 324 S 4 43 1 5 4 4 6 6 6 6 5 3 5 6 0 5 4 4 5 325 N 5 4 3 3 6 4 4 6 6 6 6 0 4 4 66 6 7 5 5 326 K 3 3 2 3 3 3 4 4 0 4 4 3 3 3 5 3 3 4 3 4 327 A 0 2 2 1 50 3 4 3 5 4 3 3 3 4 4 5 5 3 4 328 L 2 2 2 2 3 2 3 3 4 0 3 4 3 4 5 3 3 33 3 329 P 2 2 1 1 3 2 3 6 3 4 4 3 0 3 4 2 2 4 2 4 330 A 0 2 3 3 3 2 2 43 3 3 2 2 2 3 3 2 3 3 3 331 P 4 1 2 3 4 2 3 4 2 4 4 2 0 2 2 3 3 4 3 4332 I 2 0 0 0 1 1 1 0 0 2 2 0 1 0 1 1 0 1 2 1 333 E 3 3 2 0 4 3 3 4 4 54 3 2 4 4 3 3 4 3 4 334 K 0 0 0 0 4 0 1 2 0 1 1 0 0 0 1 0 0 1 2 2 335 T2 1 0 0 2 2 1 3 2 3 3 3 1 3 2 1 0 2 1 2 336 I 0 0 0 0 2 0 0 0 2 1 1 0 01 1 0 0 0 2 1 337 S 2 1 0 0 3 1 2 2 1 3 2 3 0 2 1 0 1 3 2 2 338 K 2 0 00 1 0 2 2 0 2 2 0 1 0 3 0 0 2 1 2 339 A 0 1 1 0 2 0 2 2 2 3 2 2 1 1 2 20 2 2 2 340 K 1 0 1 2 3 1 3 3 0 3 3 2 2 2 2 2 2 3 3 3 341 G 2 2 1 1 4 03 6 3 3 3 3 3 5 3 3 3 4 1 4 342 Q 4 1 1 1 5 2 4 5 5 5 5 2 2 0 4 4 2 4 54 343 P 2 1 1 1 4 2 3 1 2 4 2 3 0 2 2 2 2 2 3 2 344 R 1 1 2 1 2 1 1 1 21 1 1 0 1 0 1 1 1 3 1 345 E 3 3 2 0 4 3 2 4 3 4 4 2 3 3 3 3 3 4 4 4 346P 2 1 1 0 2 1 2 2 2 2 2 1 0 2 2 2 2 2 2 2 347 Q 3 1 1 1 3 1 2 3 2 3 3 21 0 2 2 2 2 1 3 348 V 2 2 −1 0 2 1 1 2 3 2 2 1 1 1 3 2 2 0 2 2 349 Y 2 11 1 2 2 2 2 2 2 2 3 1 2 2 1 2 2 3 0 350 T 2 2 3 2 3 2 2 4 2 4 4 3 2 3 32 0 4 3 4 351 L 0 −2 0 −2 0 −1 −1 0 −1 0 1 0 −1 0 −1 0 −1 0 0 1 352 P 22 1 1 2 2 3 2 3 3 2 3 0 2 3 3 3 2 2 2 353 P 2 0 0 −1 2 0 0 2 0 3 1 1 0 10 1 0 1 0 1 354 S 1 0 1 0 1 0 2 1 2 2 2 1 0 2 2 0 1 1 1 2 355 R 0 −1 −10 2 0 1 1 0 2 2 1 0 −1 0 0 0 2 1 1 356 K −3 −3 −3 −4 −1 −3 −1 −1 0 −1 −1−1 −3 −1 0 −3 −2 −1 −1 −1 357 E 1 0 −1 0 2 1 1 2 1 2 3 2 −1 1 1 1 1 3 22 358 M −2 −3 −3 −3 −1 −2 0 0 −1 0 0 −2 −2 −2 0 −2 −1 1 0 1 359 T 1 −1 11 2 0 2 2 2 2 2 2 −2 2 2 1 0 2 2 2 360 K 0 −1 −2 −3 1 −1 0 1 0 2 2 −1 −20 2 0 −1 2 1 1 361 N 2 1 −1 0 2 1 0 2 3 2 2 0 1 2 3 3 2 2 2 2 362 Q 1 0−2 −2 4 0 0 4 1 3 3 2 0 0 2 1 1 3 2 4 363 V 0 0 −1 −1 2 0 2 2 2 1 2 1 12 3 2 2 0 3 1 364 S 1 −1 0 0 4 0 1 4 0 4 3 2 0 0 2 0 1 4 2 4 365 L 3 1 00 2 1 3 4 2 0 4 3 2 3 2 3 1 3 2 2 366 T 5 2 2 2 5 2 3 6 1 6 6 4 1 3 3 40 5 3 5 367 C 5 0 3 2 4 4 5 4 5 4 4 4 3 4 6 4 4 4 4 4 368 L 3 2 2 2 4 24 5 3 0 2 4 2 3 4 4 3 2 2 3 369 V 2 1 1 1 5 1 1 3 2 3 3 2 1 2 3 1 1 0 22 370 K 4 3 4 4 5 3 3 5 0 5 5 3 3 4 5 5 4 6 5 5 371 G 5 5 2 4 5 0 4 6 55 5 4 3 4 6 5 5 6 6 5 372 F 2 2 2 1 0 1 4 5 4 5 5 2 2 3 5 2 4 5 3 5 373Y 6 3 4 4 4 4 4 6 4 5 6 4 3 6 5 5 4 5 3 0 374 P 5 4 3 5 7 4 6 7 5 8 7 50 5 6 8 7 7 7 7 375 S 4 2 2 2 3 2 2 3 4 3 3 3 2 3 4 0 2 3 3 3 376 D 5 40 3 4 4 5 4 4 5 5 3 4 5 5 6 5 4 3 5 377 I 2 2 2 2 4 2 4 0 2 4 3 4 2 3 32 3 3 3 3 378 A 0 2 2 2 2 2 4 3 3 3 2 3 3 4 3 0 3 3 2 3 379 V 1 1 1 1 31 2 3 1 3 0 1 1 2 1 1 1 0 3 3 380 E 2 2 1 0 4 2 2 5 3 4 5 3 2 3 3 2 2 54 5 381 W 2 1 1 1 2 1 2 1 1 1 3 1 1 1 2 1 1 1 0 3 382 E 3 1 0 0 2 1 2 31 3 3 3 1 2 3 3 3 2 2 3 383 S 0 0 0 0 0 0 1 1 0 0 1 0 0 0 0 0 0 0 0 1384 N 0 0 0 0 0 0 0 1 1 1 1 0 0 0 1 0 0 0 0 0 385 G 2 0 0 0 2 0 0 3 2 23 1 0 0 2 1 0 2 1 3 386 Q 1 1 0 1 1 1 1 1 1 1 1 0 1 0 1 1 1 1 1 1 387 P1 0 1 1 2 0 2 2 0 2 3 1 0 1 1 1 2 2 0 2 388 E 1 1 0 0 3 1 2 3 1 3 4 2 12 1 1 1 3 3 3 389 N 1 1 1 1 2 1 1 2 2 2 2 0 0 1 2 1 2 2 2 2 390 N 1 1 00 2 2 1 2 2 2 2 0 0 2 2 2 2 2 2 2 391 Y 1 0 0 1 2 1 1 2 2 3 2 1 1 2 2 11 2 2 0 392 K 2 2 2 2 3 2 3 3 0 3 3 0 0 2 3 3 2 3 3 3 393 T 2 2 1 1 1 12 2 1 3 3 3 1 2 2 3 0 3 1 1 394 T 2 1 1 1 1 2 1 2 3 2 2 2 1 2 3 2 0 2 12 395 P 3 2 1 1 3 2 1 3 2 3 3 3 0 2 2 3 3 3 2 3 396 P 2 3 0 0 3 1 3 4 14 4 2 0 1 1 3 2 4 3 3 397 V 2 2 2 2 1 2 2 3 0 3 0 3 2 2 2 3 2 0 1 1 398L 2 2 2 1 3 2 2 4 2 0 3 2 2 2 2 2 2 3 3 3 399 D 4 3 0 2 4 3 4 4 4 5 5 43 3 4 3 3 4 4 4 400 S 2 2 2 1 2 2 3 2 2 3 3 2 2 2 2 0 2 2 2 2 401 D 1 10 0 2 1 3 3 3 3 3 0 1 2 3 1 2 2 2 3 402 G 3 1 1 1 2 0 2 2 2 2 2 2 2 2 22 2 2 2 2 403 S 2 2 1 1 3 2 3 3 3 4 4 2 2 3 3 0 2 3 2 3 404 F 3 3 2 3 03 2 3 2 4 3 4 3 3 3 3 3 3 3 3 405 F 3 3 1 2 0 3 3 4 3 5 4 3 3 3 3 3 3 43 4 406 L 4 2 3 3 4 2 4 4 4 0 4 2 1 3 4 4 3 4 5 6 407 Y 5 4 2 3 7 4 4 56 5 5 6 4 6 6 5 6 6 6 0 408 S 6 4 3 3 6 5 4 5 5 6 6 6 3 5 5 0 4 5 6 5409 K 5 5 4 5 6 4 5 7 0 7 7 5 4 5 0 5 4 7 6 6 410 L 4 3 4 4 6 3 4 5 6 04 3 3 4 6 3 3 4 7 5 411 T 5 5 5 4 8 4 6 9 4 8 8 6 6 6 8 6 0 8 7 8 412 V5 4 2 4 6 4 5 5 4 6 6 4 4 4 5 4 4 0 4 5 413 D 7 7 0 3 8 6 7 8 8 7 8 7 68 9 6 7 7 7 8 414 K 3 3 3 3 5 3 4 4 0 5 5 3 2 3 3 3 4 4 4 5 415 S 3 2 11 4 1 2 4 3 3 3 2 2 2 4 0 2 2 3 3 416 R 2 1 1 1 3 1 1 3 1 3 3 2 2 2 0 11 3 2 3 417 W 2 2 2 1 3 0 2 3 2 3 3 4 3 1 3 4 2 3 0 3 418 Q 4 3 2 2 5 26 6 6 5 6 3 1 0 6 4 3 4 5 7 419 Q 1 2 1 0 2 1 1 2 4 4 3 2 1 0 5 2 1 2 33 420 G 2 2 1 2 3 0 1 3 3 3 3 4 0 3 3 4 3 3 4 3 421 N 1 3 1 1 4 1 3 5 44 5 0 2 2 7 3 3 5 4 4 422 V 1 0 1 0 4 1 2 0 2 4 4 2 1 3 4 1 1 0 4 4 423F 3 1 2 2 0 2 5 2 3 4 2 3 1 3 2 2 3 3 5 5 424 S 4 3 0 2 6 4 4 6 4 7 6 42 5 5 0 4 7 6 6 425 C 6 0 3 3 6 5 6 6 6 6 6 6 5 5 6 7 6 7 6 6 426 S 4 33 3 5 3 3 5 3 6 6 5 4 4 4 0 4 4 5 5 427 V 3 3 1 1 4 2 3 5 3 5 5 3 3 3 43 3 0 3 3 428 M 2 2 1 1 4 2 3 6 1 5 0 3 2 3 2 3 3 6 3 5 429 H 6 5 2 3 74 0 7 4 8 7 5 5 4 5 4 4 8 7 8 430 E 7 6 6 0 8 6 7 9 8 9 9 7 7 6 9 9 9 88 8 431 A 0 5 3 2 7 6 6 7 5 7 7 6 5 4 6 5 6 6 7 7 432 L 3 3 3 2 5 4 5 54 0 6 6 4 5 5 5 4 5 5 5 433 H 5 5 1 2 6 4 0 7 6 8 8 5 4 6 6 5 5 7 6 6434 N 5 5 4 4 6 5 7 7 8 8 7 0 5 5 8 6 6 7 6 6 435 R 5 5 3 2 5 4 0 6 6 65 5 2 5 0 5 5 6 5 5 436 Y 7 6 3 5 0 6 5 7 7 7 7 6 5 6 8 6 4 7 7 0 437 T7 6 4 5 8 6 7 8 8 8 8 8 7 8 8 7 0 8 8 8 438 Q 6 5 3 2 7 5 7 5 4 5 7 7 50 6 6 6 6 6 6 439 K 6 4 3 3 4 4 4 5 0 4 5 5 3 4 5 5 5 4 4 5 440 S 5 4 33 3 4 4 5 4 4 4 4 4 4 4 0 4 4 3 3 441 L 4 3 2 3 4 4 4 4 3 0 4 3 3 3 3 44 4 4 4 442 S 3 3 2 2 4 3 3 4 3 4 3 3 3 3 4 0 3 3 2 4 443 L 2 2 2 1 2 23 2 1 0 2 3 2 3 3 2 2 2 2 2 444 S 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 0 2 2 12 445 P 1 1 1 1 1 1 1 1 1 0 1 1 0 1 1 1 1 1 1 1

TABLE 68 EU BH076 No. sequence A C D E F G H I K L M N P Q R S T V W Y118 A 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 119 S 0 0 0 0 1 0 0 1 0 11 0 0 0 0 0 0 1 1 1 120 T 0 0 0 0 1 0 0 1 0 1 1 0 0 0 0 0 0 1 1 1 121 K0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 122 G 0 0 0 0 1 0 0 0 0 0 0 0 00 0 0 0 0 0 1 123 P 1 0 0 0 2 0 1 1 1 2 1 0 0 0 2 1 0 1 2 2 124 S 0 0 00 2 0 0 2 2 2 2 1 0 1 2 0 0 1 1 1 125 V 0 0 0 0 1 0 0 1 0 1 1 0 0 0 0 00 0 1 1 126 F 0 1 0 1 0 0 1 2 2 2 2 2 0 1 2 1 1 2 3 2 127 P 4 3 3 3 4 33 4 2 4 4 3 0 3 2 3 3 4 4 4 128 L 1 1 2 2 3 1 2 3 4 0 3 2 1 2 3 2 1 3 33 129 A 0 3 3 3 4 3 3 3 3 3 3 3 2 4 3 3 3 3 4 3 130 P 3 3 3 3 4 3 3 4 34 4 3 0 3 4 3 3 3 5 4 131 S 3 3 3 3 4 3 2 4 3 4 4 3 3 3 3 0 3 4 3 4 132S 3 3 3 3 4 3 3 4 3 4 4 3 3 3 3 0 3 4 4 4 133 K 3 2 3 3 3 2 3 3 0 3 3 33 3 3 3 3 3 3 3 134 S 3 3 3 3 4 3 3 4 3 4 5 3 3 3 3 0 3 4 4 4 135 T 2 22 1 3 2 2 4 2 4 4 2 2 2 3 2 0 2 3 3 136 S 1 1 1 1 2 1 1 2 1 2 3 1 1 1 10 1 1 2 2 137 G 1 1 1 1 2 0 1 2 1 3 2 1 1 1 1 1 1 2 2 2 138 G 0 1 0 0 10 0 1 1 2 1 0 0 0 1 0 0 1 1 1 139 T 1 1 0 0 3 0 1 3 1 3 3 0 0 1 1 0 0 31 3 140 A 0 0 0 0 1 0 1 1 1 1 1 0 0 0 1 0 0 1 1 1 141 A 0 0 0 0 2 0 0 20 1 1 0 0 0 0 0 0 0 2 2 142 L 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1143 G 2 0 1 1 2 0 1 2 1 2 2 1 1 2 2 1 1 2 2 2 144 C 2 0 0 1 4 2 3 5 2 55 2 2 3 3 2 1 4 4 4 145 L 0 0 1 1 0 0 1 0 1 0 0 0 0 1 1 1 0 0 1 0 146 V0 0 0 0 2 0 1 0 1 2 1 1 1 0 1 1 1 0 1 1 147 K 0 0 0 0 3 0 1 3 0 3 3 1 11 2 1 1 3 1 3 148 D 2 2 0 2 4 2 3 4 4 4 4 2 2 3 3 3 3 4 4 4 149 Y 1 0 10 1 0 2 2 1 3 3 2 1 0 3 2 1 2 2 0 150 F 2 1 2 2 0 1 3 2 3 2 1 2 2 3 4 43 2 1 2 151 P 3 2 2 2 3 2 3 4 4 5 5 3 0 4 3 3 2 2 3 3 152 E 4 2 1 0 4 24 3 3 3 3 3 2 3 4 5 2 3 4 4 153 P 3 2 3 3 3 1 3 5 2 5 5 1 0 2 4 3 2 3 34 154 V 2 1 1 1 2 1 2 2 2 2 2 1 1 2 2 2 1 0 1 2 155 T 3 2 2 2 4 2 3 4 34 4 2 2 2 3 3 0 4 4 4 156 V 2 2 1 2 3 2 2 3 2 3 3 2 2 2 3 2 2 0 3 3 157S 3 3 2 2 5 3 3 4 2 5 5 2 0 2 3 0 3 5 4 5 158 W 3 3 3 2 3 3 3 3 4 3 3 32 3 4 4 4 4 0 3 159 N 2 2 1 1 2 2 2 2 3 2 2 0 1 3 2 2 2 3 2 2 160 S 4 32 2 4 4 4 4 4 4 4 3 3 3 3 0 4 4 4 4 161 G 4 3 2 2 5 0 5 5 4 5 5 4 3 4 43 3 5 4 5 162 A 0 4 1 1 5 3 2 6 5 5 6 4 4 3 5 4 4 6 5 5 163 L 3 1 1 1 23 2 3 3 0 4 2 3 2 3 3 3 3 1 2 164 T 3 3 1 2 4 3 3 5 3 5 5 3 3 3 3 3 0 45 4 165 S 3 2 1 1 2 1 3 4 3 3 3 2 3 2 3 0 2 3 2 3 166 G 4 2 1 2 4 0 5 53 5 4 4 2 4 6 5 3 5 4 4 167 V 3 3 4 0 4 3 3 3 4 5 5 4 3 4 4 4 3 0 4 4168 H 2 1 1 1 3 2 0 3 3 3 3 2 1 1 3 1 2 2 2 3 169 T 3 3 2 1 4 3 4 5 3 44 4 2 4 5 3 0 4 4 4 170 F 3 1 1 1 0 1 1 3 3 2 3 3 2 3 2 2 2 3 1 3 171 P6 4 3 3 6 3 6 6 7 6 6 5 0 6 6 4 4 6 5 5 172 A 0 5 0 0 6 3 6 6 5 6 6 4 45 7 6 3 6 6 6 173 V 3 2 2 2 7 2 5 6 2 6 6 3 2 5 3 4 3 0 6 5 174 L 4 5 34 6 4 4 6 5 0 6 3 2 2 5 4 4 6 6 6 175 Q 5 4 5 4 5 5 7 6 7 6 6 5 4 0 7 66 5 6 6 176 S 7 6 3 4 6 6 5 6 6 6 6 5 5 5 6 0 6 6 6 0 177 S 6 3 3 4 5 45 6 5 5 5 5 3 3 5 0 5 6 6 5 178 G 8 7 5 4 8 0 8 8 8 8 8 6 7 7 9 7 7 8 88 179 L 4 6 4 2 7 5 6 7 6 0 7 6 5 7 6 6 5 7 7 7 180 Y 5 4 3 3 6 2 6 6 66 6 3 4 4 6 5 4 6 7 0 181 S 6' 6 5 3 7 5 6 7 8 7 7 7 5 7 8 0 7 7 8 8 182L 6 6 3 4 7 5 6 7 5 0 7 6 5 6 8 7 5 7 6 7 183 S 6 5 4 3 6 6 5 6 7 6 6 56 7 7 0 6 6 6 6 184 S 6 5 4 4 7 5 7 8 7 7 7 5 4 6 7 0 6 6 6 6 185 V 4 54 3 6 4 5 6 5 6 6 6 3 5 5 5 5 0 5 6 186 V 4 4 4 5 5 4 5 5 5 5 5 4 4 4 55 4 0 5 6 187 T 6 5 6 5 8 5 6 8 6 8 8 5 5 7 6 5 0 8 7 8 188 V 4 3 4 4 44 4 4 4 4 4 3 3 3 5 4 4 0 4 4 189 P 6 4 4 4 5 4 5 7 6 7 7 4 0 5 6 4 3 56 7 190 S 2 3 2 2 3 2 2 3 2 3 3 2 2 3 2 0 2 3 2 3 191 S 2 2 2 2 3 2 2 33 4 3 2 2 2 4 0 2 3 3 2 192 S 2 1 1 1 3 1 3 3 2 3 3 2 1 2 3 0 1 3 2 3193 L 1 1 2 1 2 1 2 2 2 0 1 3 1 2 2 1 2 1 2 2 194 G 3 2 1 2 6 0 3 6 4 66 3 2 3 4 2 1 5 5 6 195 T 1 0 0 0 4 0 3 4 2 4 5 1 0 3 2 0 0 3 2 4 196 02 1 1 0 3 2 1 3 2 4 3 2 1 0 4 2 1 3 3 3 197 T 1 1 1 1 2 1 1 5 1 3 3 2 01 2 1 0 3 4 3 198 Y 2 1 0 0 2 2 1 3 2 3 4 2 2 3 2 1 2 3 2 0 199 I 2 1 12 3 2 2 0 2 3 3 2 1 2 2 3 0 3 3 3 200 C 5 0 2 3 6 4 6 7 6 6 6 5 6 6 6 55 7 4 6 201 N 5 3 3 3 5 4 5 4 4 5 5 0 1 4 5 4 4 3 4 5 202 V 4 3 2 2 5 36 5 5 6 5 5 2 6 5 4 3 0 4 5 203 N 4 3 3 2 5 2 5 6 5 5 5 0 3 3 6 5 5 5 45 204 H 5 3 4 4 5 5 0 6 5 6 6 5 3 6 4 5 3 6 4 5 205 K 4 4 2 4 5 4 4 6 06 5 5 2 4 5 4 4 5 5 5 206 P 5 3 2 2 5 4 3 5 5 6 5 6 0 5 6 5 5 5 4 5 207S 4 3 3 2 4 2 4 5 3 4 4 3 4 4 5 0 4 4 3 4 208 N 3 2 2 2 4 3 2 4 2 5 4 03 3 3 2 2 4 5 4 209 T 2 2 1 1 3 2 2 3 3 3 3 3 2 2 3 2 0 3 2 3 210 K 2 11 1 1 1 1 1 0 2 1 1 1 1 1 1 1 1 2 1 211 V 2 1 1 1 1 1 2 2 2 2 1 2 2 2 21 1 0 1 1 212 D 2 1 0 0 4 1 2 3 1 3 3 1 1 2 3 1 1 3 2 3 213 K 0 0 0 0 20 2 2 0 1 1 1 0 1 1 0 1 1 2 2 214 K 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 10 0 215 V 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 216 E 1 1 0 0 2 1 1 32 3 2 1 1 2 2 1 2 3 1 2 217 P 0 0 0 0 2 0 1 2 1 2 2 0 0 1 1 0 0 2 2 2218 K 0 0 0 0 2 0 0 2 0 2 2 0 0 1 0 1 0 2 2 2 219 S 1 0 0 0 0 0 0 1 0 10 0 0 1 0 0 0 0 0 0 220 C 0 0 0 0 0 0 1 1 2 1 1 1 1 1 3 2 1 0 1 1 221 D2 0 0 0 3 2 1 1 1 2 3 2 0 1 2 1 0 1 3 3 222 K 0 0 0 0 0 0 1 1 0 0 0 1 01 0 0 0 0 0 0 223 T 0 0 0 0 2 0 0 0 0 0 0 0 0 1 1 1 0 0 1 1 224 H 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 225 T 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 00 0 1 1 226 C 0 0 0 0 1 0 0 1 0 0 0 1 0 0 0 0 0 0 1 1 227 P 0 1 0 0 1 10 1 1 1 1 1 0 1 1 1 0 1 1 1 228 P 0 0 0 0 2 0 1 0 0 0 0 0 0 0 0 0 0 0 11 229 C 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 230 P 0 0 0 0 0 0 0 1 01 1 0 0 0 0 0 0 1 0 0 231 A 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 232P 1 1 1 0 1 1 2 1 1 1 1 1 0 1 1 1 1 1 1 1 233 E 1 1 1 0 3 1 2 3 1 3 3 20 1 2 2 1 3 2 2 234 L 1 1 0 0 0 1 2 2 2 0 2 0 0 0 2 1 1 2 2 2 235 L 1 11 1 1 1 1 2 1 0 2 1 1 1 1 1 1 1 1 1 236 G 2 2 1 2 3 0 1 2 2 2 2 2 2 2 22 2 2 3 3 237 G 2 1 1 1 1 0 1 1 1 1 1 2 2 2 1 2 1 1 1 1 238 P 3 1 2 1 41 3 4 2 4 4 3 0 3 3 3 2 4 4 4 239 S 2 3 1 1 3 3 2 3 3 3 3 2 2 2 3 0 3 33 3 240 V 2 1 1 1 3 1 2 4 4 4 4 1 1 2 3 1 1 0 2 3 241 F 3 1 1 1 0 1 2 54 5 5 1 1 1 3 1 1 5 4 5 242 L 2 2 2 1 2 2 2 3 2 0 3 2 2 3 3 2 2 2 2 2243 F 4 3 2 2 0 2 4 4 4 4 4 3 3 4 4 5 4 4 2 4 244 P 2 1 2 2 3 2 3 3 3 33 4 0 3 5 3 3 3 3 3 245 P 4 1 1 0 4 3 4 4 3 5 4 3 0 2 3 3 2 4 4 4 246 K2 0 2 1 2 1 2 3 0 4 3 1 2 1 3 2 2 3 3 3 247 P 2 2 1 1 3 2 2 3 2 4 4 2 02 4 2 2 2 2 3 248 K 3 1 2 1 4 2 4 4 0 4 4 1 1 3 4 3 2 4 4 4 249 D 4 4 03 5 4 4 5 6 5 5 4 3 4 6 4 4 5 6 6 250 T 2 2 2 2 2 2 2 2 3 3 3 2 2 2 3 20 2 3 2 251 L 2 2 1 1 3 1 2 2 2 0 3 3 1 1 3 3 3 2 2 3 252 M 3 3 1 2 3 33 3 3 3 0 3 3 3 4 3 3 3 3 4 253 I 4 3 3 2 3 2 3 0 4 3 3 4 3 3 4 4 4 3 33 254 S 4 3 3 3 4 3 3 4 3 4 4 4 3 4 3 0 4 4 4 4 255 R 3 2 2 2 4 2 4 5 34 4 3 3 3 0 4 3 4 3 3 256 T 3 3 3 3 5 3 3 5 3 5 5 3 3 4 3 4 0 5 5 5 257P 4 3 3 2 5 4 4 5 3 5 5 4 0 5 3 4 4 5 5 5 258 E 4 3 3 0 4 3 3 4 5 4 5 44 3 4 4 4 5 4 4 259 V 3 3 3 3 4 3 3 4 3 4 4 3 3 3 4 3 3 0 4 4 260 T 3 33 3 4 3 3 4 4 4 4 3 2 3 4 3 0 4 4 4 261 C 3 0 1 1 4 2 3 5 3 4 4 2 2 3 32 2 4 3 4 262 V 0 1 0 0 1 0 1 2 1 1 1 1 0 1 1 1 1 0 2 1 263 V 2 1 1 1 11 1 1 1 1 1 1 0 1 1 1 1 0 1 1 264 V 1 1 1 1 1 0 1 1 1 1 1 1 1 1 2 1 1 00 1 265 D 3 2 0 1 3 2 2 3 2 3 3 2 1 3 3 3 2 3 3 3 266 V 1 1 0 0 2 1 2 12 1 1 2 1 1 2 1 1 0 1 2 267 S 0 2 2 1 2 0 2 2 1 2 2 2 1 2 3 0 1 2 1 2268 H 1 0 1 0 2 2 0 3 1 3 3 1 3 1 2 2 1 3 2 2 269 E 3 1 1 0 3 2 2 3 2 33 2 2 2 3 2 2 3 3 3 270 E 1 0 0 0 1 0 2 2 1 2 2 0 1 0 2 1 0 1 0 1 271 P0 0 0 0 1 0 1 1 0 1 1 1 0 1 0 0 0 1 1 0 272 E 0 0 0 0 2 0 0 2 0 1 2 0 00 2 0 0 1 1 2 273 V 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 274 K 1 1 00 2 1 1 1 0 1 1 0 0 0 1 1 1 0 1 2 275 F 0 0 0 0 0 0 0 1 1 1 1 0 0 0 0 00 1 1 1 276 N 1 1 1 1 3 1 2 3 1 3 3 0 1 2 2 2 2 3 3 3 277 W 1 1 0 0 2 11 2 2 2 2 1 0 1 2 2 2 2 0 2 278 Y 2 1 1 0 2 1 2 2 3 3 2 2 2 3 3 3 3 3 20 279 V 2 2 1 1 2 2 2 2 2 2 2 2 2 3 2 2 2 0 2 2 280 D 4 3 0 2 6 3 4 5 65 5 3 2 5 6 4 4 5 5 7 281 G 3 3 2 2 3 0 3 4 3 5 5 3 2 3 4 3 2 3 3 3 282V 3 2 1 2 2 2 3 2 3 2 0 2 3 2 4 3 2 0 2 2 283 E 4 3 2 0 6 4 3 6 5 7 6 44 4 7 4 4 7 5 6 284 V 3 2 1 1 3 2 2 3 3 4 3 4 2 2 2 2 2 0 3 3 285 H 4 32 2 4 3 0 4 3 4 4 3 2 4 3 3 2 4 3 4 286 N 3 3 2 2 4 3 3 4 3 5 4 0 3 3 43 3 3 4 4 287 A 0 2 2 1 2 2 2 2 3 2 2 2 1 2 3 2 1 2 3 3 288 K 1 0 0 0 21 3 3 0 3 3 3 2 1 2 2 2 3 2 2 289 T 2 1 2 2 3 2 4 3 4 3 3 3 1 3 4 2 0 32 3 290 K 1 1 1 0 3 1 2 4 0 4 3 2 2 2 2 1 3 3 3 4 291 P 1 2 1 2 5 1 3 22 0 3 1 0 2 3 2 2 2 1 3 292 R 1 2 1 1 3 1 2 3 1 3 3 1 1 1 0 2 1 3 1 3293 E 2 1 2 0 4 2 2 4 3 3 4 2 2 2 3 2 3 4 4 4 294 E 3 3 1 0 4 2 3 5 4 54 2 4 4 4 4 3 5 3 4 295 Q 0 0 0 0 1 0 0 2 0 2 2 0 0 0 0 0 0 2 1 2 296 Y0 0 0 0 0 0 1 2 2 1 1 0 0 0 1 1 0 1 2 0 297 N 3 2 1 1 3 1 1 3 3 3 3 0 11 2 2 1 3 3 3 298 S 2 1 1 1 4 1 1 4 4 4 4 2 1 2 4 0 1 4 3 4 299 T 3 4 22 3 3 3 4 5 4 4 3 3 2 5 2 0 3 3 3 300 Y 5 3 1 2 0 4 3 3 3 4 4 4 3 4 5 55 4 3 0 301 R 4 4 1 2 5 3 4 4 3 5 4 3 4 4 0 3 3 4 5 5 302 V 5 4 3 3 4 44 4 4 4 4 5 3 4 5 5 4 0 4 4 303 V 4 4 4 3 5 4 4 6 6 6 6 5 4 4 4 4 5 0 76 304 S 5 5 4 4 6 5 6 8 6 6 6 5 5 8 8 0 5 7 6 6 305 V 5 5 4 5 6 5 5 6 56 7 5 5 5 5 5 5 0 6 6 306 L 7 6 3 3 7 6 6 7 7 0 7 6 6 6 7 6 6 7 6 7 307T 7 6 5 5 8 6 7 7 6 8 8 6 7 6 6 6 0 7 8 7 308 V 6 6 5 5 7 5 7 7 5 8 8 65 6 5 6 6 0 6 7 309 L 5 4 4 4 6 5 4 7 5 0 6 6 6 5 6 7 6 0 5 6 310 H 5 33 2 6 4 0 6 5 6 5 4 5 7 6 6 5 5 5 6 311 Q 4 4 3 4 5 4 4 5 4 5 5 4 4 0 64 4 4 5 5 312 D 4 5 0 2 6 4 5 7 5 8 6 5 4 4 6 5 5 6 7 6 313 W 3 1 1 1 33 4 3 2 3 3 2 2 1 3 3 3 3 0 3 314 L 2 2 2 2 3 2 3 3 4 0 3 3 3 2 4 2 4 33 3 315 N 2 2 1 1 2 2 3 4 2 4 3 0 2 2 3 3 2 3 2 2 316 G 1 1 0 0 3 0 1 20 3 2 2 0 2 2 0 0 2 2 4 317 K 0 0 −1 −1 1 0 1 1 0 1 0 0 0 0 3 1 0 0 1 1318 E 1 0 1 0 2 1 1 3 1 3 3 1 1 1 2 2 3 3 3 3 319 Y −1 −1 −1 −1 0 −1 −10 0 0 0 −1 −1 −1 0 −1 −1 0 0 0 320 K 1 −1 −2 −2 1 1 0 1 0 1 1 1 −1 1 1−1 −1 1 0 1 321 C 0 0 0 0 2 0 1 1 1 1 1 0 0 0 2 1 0 1 1 2 322 K 1 −1 −1−1 1 0 1 2 0 2 2 0 0 0 1 0 1 2 1 2 323 V 0 0 0 0 0 0 −1 0 0 0 0 1 0 0 00 0 0 0 0 324 S 1 1 −2 −2 1 0 0 1 2 1 2 0 0 −1 3 0 0 2 1 1 325 N 1 0 −10 1 1 1 1 1 1 1 0 0 1 1 1 1 1 2 1 326 D 1 1 0 1 1 1 2 1 0 1 1 3 1 1 3 12 1 1 1 327 A 0 0 −1 −1 1 0 1 0 1 0 0 0 −1 0 1 1 0 0 0 0 328 L 0 −1 −1−1 0 0 0 0 1 0 1 2 1 1 2 0 2 0 0 1 329 P 0 0 0 0 2 0 0 2 0 2 2 0 0 0 0 10 2 2 2 330 M −1 −1 0 0 0 −1 −1 0 −1 1 0 −1 −1 −1 −1 −1 −1 0 0 0 331 P 11 1 2 3 1 2 3 2 3 4 1 0 1 2 1 1 2 3 3 332 I −1 −1 −1 −1 0 −1 0 0 0 0 0−1 0 0 0 0 0 0 0 0 333 E 0 0 0 0 1 0 0 1 0 1 1 0 −1 1 0 0 −1 1 2 1 334 E0 0 0 0 3 0 1 1 0 0 0 0 0 0 1 0 0 0 1 1 335 T −1 0 −1 −1 0 0 1 0 1 0 0 10 1 1 −1 0 0 0 0 336 I 0 −1 0 0 1 0 −1 0 0 0 0 0 0 1 0 0 −1 0 1 1 337 S2 1 −1 0 3 1 1 2 1 2 2 1 0 1 1 0 1 3 2 2 338 K 1 0 0 0 1 0 1 1 0 1 1 0 10 2 0 0 1 1 1 339 A 0 1 0 0 2 0 2 2 2 2 2 1 0 1 2 2 0 2 2 2 340 K 0 0 11 2 0 1 2 0 2 2 1 1 1 1 1 1 2 2 2 341 G 2 1 0 0 3 0 2 4 2 2 2 2 2 4 2 22 3 1 3 342 Q 3 1 1 1 3 1 3 3 4 3 3 2 2 0 3 3 2 2 4 3 343 P 2 1 1 1 3 23 1 2 3 2 3 0 2 2 2 2 2 3 2 344 R 1 1 2 1 2 1 1 1 2 1 1 1 0 1 0 1 1 1 31 345 E 3 3 2 0 4 3 2 4 3 4 4 2 3 3 3 3 3 4 4 4 346 P 2 1 1 0 2 1 2 2 22 2 1 0 2 2 2 2 2 2 2 347 Q 2 1 1 1 3 1 1 3 1 3 3 2 1 0 1 2 2 2 1 3 348V 3 2 0 1 3 2 3 3 4 3 3 2 2 3 4 2 2 0 3 3 349 Y 3 2 2 2 3 3 3 3 3 3 3 42 3 3 2 3 3 3 0 350 T 3 3 3 3 3 3 3 3 3 3 3 4 3 3 3 3 0 3 3 3 351 L 2 12 0 2 0 2 2 1 0 2 2 1 2 1 2 2 2 2 2 352 P 3 2 2 2 3 2 3 3 3 3 3 3 0 2 33 2 3 3 3 353 P 2 2 2 0 2 2 2 2 2 2 2 2 0 2 1 2 2 2 2 2 354 S 2 2 2 2 22 2 2 2 2 3 2 2 2 2 0 2 1 1 2 355 R 2 2 2 3 3 2 2 4 2 3 3 2 2 0 0 2 2 33 3 356 E 1 1 1 0 3 1 3 3 4 3 3 3 1 3 4 1 2 3 3 3 357 E 2 1 1 0 3 2 2 42 4 4 2 1 2 1 2 2 4 3 3 358 M 0 0 0 0 2 0 0 1 1 0 0 0 0 0 1 0 0 0 2 2359 T 1 0 1 1 2 1 2 2 2 2 2 2 0 2 2 1 0 2 2 2 360 K 1 1 0 0 2 0 1 2 0 33 0 0 1 2 0 1 3 2 2 361 N 3 2 1 1 3 2 1 4 3 4 3 0 1 4 3 3 2 4 3 3 362 Q2 2 0 0 3 1 2 4 2 4 4 2 2 0 2 2 1 4 3 3 363 V 2 1 1 1 2 2 3 2 4 1 3 2 13 4 3 4 0 4 2 364 S 1 0 0 0 4 1 2 4 1 4 3 4 1 2 3 0 2 4 2 4 365 L 3 2 01 2 1 3 4 3 0 4 3 2 3 3 3 1 3 2 2 366 T 5 2 2 2 5 2 3 6 1 6 6 4 1 3 3 40 5 3 5 367 C 5 0 3 2 4 4 5 4 5 4 4 4 3 4 6 4 4 4 4 4 368 L 3 2 2 2 4 24 5 3 0 2 4 2 3 4 4 3 2 2 3 369 V 2 1 1 1 5 1 1 3 2 3 3 2 1 2 3 1 1 0 22 370 K 4 3 4 4 5 3 3 5 0 5 5 3 3 4 5 5 4 6 5 5 371 G 5 5 2 4 5 0 4 6 55 5 4 3 4 6 5 5 6 6 5 372 F 2 2 2 1 0 1 4 5 4 5 5 2 2 3 5 2 4 5 3 5 373Y 6 3 4 4 4 4 4 6 4 5 6 4 3 6 5 5 4 5 3 0 374 P 5 4 3 5 7 4 6 7 5 8 7 50 5 6 8 7 7 7 7 375 S 4 2 2 2 3 2 2 3 4 3 3 3 2 3 4 0 2 3 3 3 376 D 5 40 3 4 4 5 4 4 5 5 3 4 5 5 6 5 4 3 5 377 I 2 2 2 2 4 2 4 0 2 4 3 4 2 3 32 3 3 3 3 378 A 0 2 2 2 2 2 4 3 3 3 2 3 3 4 3 0 3 3 2 3 379 V 1 1 1 1 31 2 3 1 3 0 1 1 2 1 1 1 0 3 3 380 E 2 2 1 0 4 2 2 5 3 4 5 3 2 3 3 2 2 54 5 381 W 2 1 1 1 2 1 2 1 1 1 3 1 1 1 2 1 1 1 0 3 382 E 3 1 0 0 2 1 2 31 3 3 3 1 2 3 3 3 2 2 3 383 S 0 0 0 0 0 0 1 1 0 0 1 0 0 0 0 0 0 0 0 1384 N 0 0 0 0 0 0 0 0 1 1 1 1 0 0 0 1 0 0 0 0 385 G 2 0 0 0 2 0 0 3 2 23 1 0 0 2 1 0 2 1 3 386 Q 1 1 0 1 1 1 1 1 1 1 1 1 0 1 0 1 1 1 1 1 387 P1 0 1 1 2 0 2 2 0 2 3 1 0 1 1 1 2 2 0 2 388 E 1 1 0 0 3 1 2 3 1 3 4 2 12 1 1 1 3 3 3 389 N 1 1 1 1 2 1 1 2 2 2 2 0 0 1 2 1 2 2 2 2 390 N 1 1 00 2 2 1 2 2 2 2 0 0 2 2 2 2 2 2 2 391 Y 1 0 0 1 2 1 1 2 2 3 2 1 1 2 2 11 2 2 0 392 K 2 2 2 2 3 2 3 3 0 3 3 0 0 2 3 3 2 3 3 3 393 T 2 2 1 1 1 12 2 1 3 3 3 1 2 2 3 0 3 1 1 394 T 2 1 1 1 1 2 1 2 3 2 2 2 1 2 3 2 0 2 12 395 P 3 2 1 1 3 2 1 3 2 3 3 3 0 2 2 3 3 3 2 3 396 P 2 3 0 0 3 1 3 4 14 4 2 0 1 1 3 2 4 3 3 397 V 2 2 2 2 1 2 2 3 0 3 0 3 2 2 2 3 2 0 1 1 398L 2 2 2 1 3 2 2 4 2 0 3 2 2 2 2 2 2 3 3 3 399 D 4 3 0 2 4 3 4 4 4 5 5 43 3 4 3 3 4 4 4 400 S 2 2 2 1 2 2 3 2 2 3 3 2 2 2 2 0 2 2 2 2 401 D 1 10 0 2 1 3 3 3 3 3 0 1 2 3 1 2 2 2 3 402 G 3 1 1 1 2 0 2 2 2 2 2 2 2 2 22 2 2 2 2 403 S 2 2 1 1 3 2 3 3 3 4 4 2 2 3 3 0 2 3 2 3 404 F 3 3 2 3 03 2 3 2 4 3 4 3 3 3 3 3 3 3 3 405 F 3 3 1 2 0 3 3 4 3 5 4 3 3 3 3 3 3 43 4 406 L 4 2 3 3 4 2 4 4 4 0 4 2 1 3 4 4 3 4 5 6 407 Y 5 4 2 3 7 4 4 56 5 5 6 4 6 6 5 6 6 6 0 408 S 6 4 3 3 6 5 4 5 5 6 6 6 3 5 5 0 4 5 6 5409 K 5 5 4 5 6 4 5 7 0 7 7 5 4 5 0 5 4 7 6 6 410 L 4 3 4 4 6 3 4 5 6 04 3 3 4 6 3 3 4 7 5 411 T 5 5 5 4 6 4 6 9 4 8 8 6 6 6 8 6 0 6 7 6 412 V5 4 2 4 6 4 5 5 4 6 6 4 4 4 5 4 4 0 4 5 413 D 7 7 0 3 8 8 7 8 8 7 8 r 68 9 6 7 7 7 8 414 K 3 3 3 3 5 3 4 4 0 5 5 3 2 3 3 3 4 4 4 5 415 S 3 2 11 4 1 2 4 3 3 3 2 2 2 4 0 2 2 3 3 416 R 2 1 1 1 3 1 1 3 1 3 3 2 2 2 0 11 3 2 3 417 W 2 2 2 1 3 0 2 3 2 3 3 4 3 1 3 4 2 3 0 3 418 Q 4 3 2 2 5 26 6 6 5 6 3 1 0 6 4 3 4 5 7 419 Q 1 2 1 0 2 1 1 2 4 4 3 2 1 0 5 2 1 2 33 420 G 2 2 1 2 3 0 1 3 3 3 3 4 0 3 3 4 3 3 4 3 421 N 1 3 1 1 4 1 3 5 44 5 0 2 2 7 3 3 5 4 4 422 V 1 0 1 0 4 1 2 0 2 4 4 2 1 3 4 1 1 0 4 4 423F 3 1 2 2 0 2 5 2 3 4 2 3 1 3 2 2 3 3 5 5 424 S 4 3 0 2 6 4 4 6 4 7 6 42 5 5 0 4 7 6 6 425 C 6 0 3 3 6 5 6 6 6 6 6 6 5 5 6 7 6 7 6 6 426 S 4 33 3 5 3 4 5 4 6 6 5 4 5 5 0 4 4 5 5 427 V 3 3 1 1 5 2 3 5 3 5 5 3 3 3 43 3 0 4 4 428 M 2 2 1 1 4 2 3 6 1 5 0 3 2 3 2 3 3 6 3 5 429 H 5 5 1 3 73 0 7 4 8 7 5 5 4 4 4 4 8 7 7 430 E 7 5 5 0 9 5 7 9 7 9 9 6 7 5 8 8 8 97 9 431 A 0 4 2 2 6 4 5 6 4 6 6 4 3 2 5 4 6 6 6 6 432 L 1 2 1 0 3 1 2 31 0 4 3 2 1 1 3 2 5 3 3 433 H 3 1 0 1 4 2 0 4 3 5 5 3 2 2 3 3 3 4 4 4434 N 2 2 1 1 4 2 2 4 3 5 5 0 2 3 3 2 4 3 4 4 435 H 2 3 0 0 3 2 0 4 3 44 2 0 3 1 2 2 4 3 4 436 Y −1 1 1 0 2 −1 2 3 2 3 3 2 0 1 4 1 1 2 1 0 437T 0 1 0 0 2 0 2 3 3 2 1 0 0 0 4 1 0 2 3 2 438 Q 1 1 0 −1 2 1 2 2 0 2 2 03 0 1 1 1 3 2 2 439 E 2 2 0 0 2 1 2 2 −2 2 2 2 −1 2 2 2 2 2 2 2 440 S −20 0 −1 −1 −2 −1 1 −1 0 0 0 0 −1 0 0 0 −1 −2 −1 441 L 2 1 1 1 −1 1 1 0 10 1 1 0 2 1 1 2 1 −1 0 442 S 0 0 −1 −1 2 −1 0 2 −1 1 1 −1 0 1 0 0 −1 0−1 −1 443 L 0 0 0 0 0 −1 0 0 −1 0 0 0 0 0 −1 0 0 0 −1 0 444 S 0 −1 0 0 00 0 0 −1 0 0 0 0 0 0 0 0 0 0 0 445 P 0 0 0 0 0 0 1 1 1 0 0 1 0 1 1 0 0 01 0

By the above-described methods and a combination thereof, variants ofH240-AK072 and H240-BH076 described below were designed (Tables 69 and70) regarding sites of residue substitution to alter the isoelectricpoint.

TABLE 69 VARIANT NAME TEMPLATE MUTATED RESIDUE IN VARIANT (DIFFERENCEFROM TEMPLATE SEQUENCE) H240-AK072 H240-G1d L234Y L235Y G236W H268DD270E S298A D356K L358M H435R H240-FA001 H240-AK072 A231K — — — — — — —— H240-FA002 H240-AK072 L242K — — — — — — — — H240-FA003 H240-AK072E233V — — — — — — — — H240-FA004 H240-AK072 A231K E233V L242K — — — — —— H240-FA005 H240-AK072 A231K E233V — — — — — — — H240-FA006 H240-AK072A231K L242K — — — — — — — H240-FA007 H240-AK072 E233V L242K — — — — — —— H240-FA008 H240-AK072 A231K E233V S239M — — — — — — H240-FA009H240-AK072 Q196K — — — — — — — — H240-FA010 H240-AK072 Q196K I199T — — —— — — — H240-FA011 H240-AK072 Q196K V263K — — — — — — — H240-FA012H240-AK072 Q196K E272K — — — — — — — H240-FA013 H240-AK072 Q196K G316K —— — — — — — H240-FA014 H240-AK072 Q196K L358K — — — — — — — H240-FA015H240-AK072 Q196K S364K — — — — — — — H240-FA016 H240-AK072 Q196K S383K —— — — — — — H240-FA017 H240-AK072 Q196K P387K — — — — — — — H240-FA018H240-AK072 Q196K V397K — — — — — — — H240-FA019 H240-AK072 L358K V397K —— — — — — — H240-FA020 H240-AK072 Q196K I199T L358K — — — — — —H240-FA021 H240-AK072 Q196K I199T L358K V397K — — — — — H240-FA022H240-AK072 Q196K I199T L358K S383K V397K — — — — H240-FA023 H240-AK072L242K — — — — — — — — H240-FA024 H240-AK072 Q196K I199T E272K L358K — —— — — H240-FA025 H240-AK072 Q196K I199T E272K L358K 5383K — — — —H240-FA026 H240-AK072 Q196K L234L E272K — — — — — — H240-FA027H240-AK072 Q196K L235L E272K — — — — — — H240-FA028 H240-AK072 Q196KL234L L235L E272K — — — — — H240-FA029 H240-AK072 Q196K P232K E272K — —— — — — H240-FA030 H240-AK072 Q196K I199T E272K — — — — — —

TABLE 70 VARIANT NAME TEMPLATE MUTATED RESIDUE IN VARIANT (DIFFERENCEFROM TEMPLATE SEQUENCE) H240-BH076 H240-G1d D270E K326D A330M K334ED356E L358M K439E — — — — — H240-FB001 H240-BH076 K274Q — — — — — — — —— — — H240-FB010 H240-BH076 A231E K274Q — — — — — — — — — — H240-FB015H240-BH076 G137E K274Q — — — — — — — — — — H240-FB016 H240-BH076 N203DK274Q — — — — — — — — — — H240-FB017 H240-BH076 G137E N203D K274Q — — —— — — — — — H240-FB018 H240-BH076 S131C K133R G137E G138S K274Q — — — —— — — H240-FB019 H240-BH076 S131C K133R G137E G138S N203D K274Q — — — —— — H240-FB020 H240-BH076 K147E K274Q — — — — — — — — — — H240-FB021H240-BH076 K274Q K288E — — — — — — — — — — H240-FB022 H240-BH076 K274QK317E — — — — — — — — — — H240-FB023 H240-BH076 K274Q K320E — — — — — —— — — — H240-FB024 H240-BH076 K274Q G341E — — — — — — — — — — H240-FB025H240-BH076 K274Q K360E — — — — — — — — — — H240-FB028 H240-BH076 N203DK274Q K288E — — — — — — — — — H240-FB029 H240-BH076 N203D K274Q K317E —— — — — — — — — H240-FB030 H240-BH076 N203D K274Q K320E — — — — — — — —— H240-FB031 H240-BH076 N203D K274Q G341E — — — — — — — — — H240-FB032H240-BH076 N203D K274Q K360E — — — — — — — — — H240-FB033 H240-BH076G138E K274Q — — — — — — — — — — H240-FB034 H240-BH076 T139E K274Q — — —— — — — — — — H240-FB035 H240-BH076 Y198E K274Q — — — — — — — — — —H240-FB036 H240-BH076 K274Q K320E — — — — — — — — — — H240-FB037H240-BH076 K274Q S324E — — — — — — — — — — H240-FB038 H240-BH076 K274QT335E — — — — — — — — — — H240-FB039 H240-BH076 K274Q S337D — — — — — —— — — — H240-FB040 H240-BH076 K274Q L358E — — — — — — — — — — H240-FB041H240-BH076 K274Q Y278E — — — — — — — — — — H240-FB042 H240-BH076 K274QK290E — — — — — — — — — — H240-FB043 H240-BH076 K274Q G316E — — — — — —— — — — H240-FB044 H240-BH076 K274Q K340E — — — — — — — — — — H240-FB045H240-BH076 K274Q Q362E — — — — — — — — — — H240-FB046 H240-BH076 K274QS383E — — — — — — — — — — H240-FB047 H240-BH076 K274Q N384E — — — — — —— — — — H240-FB048 H240-BH076 K274Q G385E — — — — — — — — — — H240-FB049H240-BH076 K274Q Q386E — — — — — — — — — — H240-FB050 H240-BH076 K274QN390E — — — — — — — — — — H240-FB051 H240-BH076 K274Q V422E — — — — — —— — — — H240-FB052 H240-BH076 K214T K274Q — — — — — — — — — — H240-FB053H240-BH076 K274E — — — — — — — — — — — H240-FB054 H240-BH076 G137E N203DK214T K274Q — — — — — — — — H240-FB055 H240-BH076 G137E N203D K214TK274Q K288E — — — — — — — H240-FB056 H240-BH076 G137E K147E N203D K274QK288E — — — — — — — H240-FB057 H240-BH076 G137E Y198E N203D K274Q K288E— — — — — — — H240-FB058 H240-BH076 G137E G138S S192N L193F I199T N203DK274Q — — — — — H240-FB059 H240-BH076 G138E K147E K274Q K288E — — — — —— — — H240-FB060 H240-BH076 G138E Y198E K274Q K288E — — — — — — — —H240-FB061 H240-BH076 G138E K214T K274Q K288E — — — — — — — — H240-FB062H240-BH076 GI38E K147E Y198E K274Q — — — — — — — — H240-FB063 H240-BH076G138E K147E K214T K274Q — — — — — — — — H240-FB064 H240-BH076 G137EG138S S192N L193F I199T N203D K214T K274Q — — — — H240-FB065 H240-BH076G137E G138S S192N L193F I199T N203D K214T K274Q K288E — — — H240-FB066H240-BH076 G137E G138S S192H L193F I199T N203D K214T K274Q N384E — — —H240-FB067 H240-BH076 G137E G138S S192N L193F I199T N203D K214T K274QN390E — — — H240-FB068 H240-BH076 G137E G138S S192N L193F I199T N203DK214T K274Q V422E — — — H240-FB069 H240-BH076 G138E K147E K274Q K288EL358E — — — — — — — H240-FB070 H240-BH076 G138E K147E K274Q K288E L358EN384E — — — — — — H240-FB071 H240-BH076 G138E K147E K274Q K288E L358EN390E — — — — — — H240-FB072 H240-BH076 G138E K147E K274Q K288E L358EV422E — — — — — — H240-FB073 H240-BH076 G138E K147E K274Q K288E L358EN390E V422E — — — — — H240-FB074 H240-BH076 G138E K147E K274Q K288EK340E N384E — — — — — — H240-FB075 H240-BH076 G137E K147E N203D K274QK288E K340E N384E — — — — — H240-FB076 H240-BH076 G138E K147E K274QK288E K338E N384E — — — — — — H240-FB077 H240-BH076 G137E K147E N203DK274Q K288E K338E N384E — — — — — H240-FB078 H240-BH076 G137E G138SK147E S192N L193F I199T N203D K214T K274Q K288E K338E N384E H240-FB079H240-BH076 G137E G138S K147E S192N L193F I199T N203D K214T K274Q K288EK338E N384E H240-FB080 H240-BH076 G137E K147E N203D K274Q K288E K338E —— — — — — H240-FB081 H240-BH076 G138E K147E K274Q K288E K338E — — — — —— — H240-FB082 H240-BH076 G138E K147E K274Q K288E N384E — — — — — — —H240-FB083 H240-BH076 G137E G138S K147E S192N L193F I199T N203D K214TK274Q K288E — — H240-FB084 H240-BH076 G137E G138S K147E S192N L193FI199T N203D K214T K274Q K288E N384E —[Construction of Expression Vector for H240-AK072/H240-BH076/L73-k0Altered Antibodies]

First, in order to construct cDNAs for modified antibodyH240-AK072/H240-BH076/L73-k0, synthetic oligo DNAs were designed usingH240-AK072 or H240-BH076 as a template in such a way that they result ina mutation at each of selected amino acid residues. Then, animal cellexpression vectors carrying genes of interest were constructed usingeach synthetic oligo DNA according to the method described in ReferenceExample 1.

[Expression and Purification of H240-AK072/H240-BH076/L73-k0 AlteredAntibody]

To assess altered antibodies of H240-AK072/H240-BH076/L73-k0, modifiedantibodies were prepared according to the method described in ReferenceExample 1 by co-expressing in given combinations an L chain (L73-k0, SEQID NO: 106) and each H chain resulting from introduction of analteration into H240-AK072 or H240-BH076 (the H chain resulting fromintroduction of an alteration into H240-AK072 is referred to as the Achain, while the H chain resulting from introduction of an alterationinto H240-BH076 is referred to as the B chain) so that each of theresulting antibodies has A chain and B chain in any of the combination.SEQ ID NOs of representative A chains and B chains are shown in Table71.

TABLE 71 NAME SEQ ID NO A H240-FA009 138 CHAIN H240-FA012 139 H240-FA020140 H240-FA021 141 H240-FA024 142 H240-FA030 143 B H240-FB001 144 CHAINH240-FB015 145 H240-FB016 146 H240-FB017 147 H240-FB020 148 H240-FB021149 H240-FB033 150 H240-FB047 151 H240-FB052 152 H240-FB056 153H240-FB059 154 H240-FB064 155 H240-FB065 156 H240-FB082 157 H240-FB083158 H240-FB084 159

[Example 28] Physicochemical Assessment of H240-AK072/H240-BH076/L73-k0Variants

[Measurement of Retention Time Difference by Cation ExchangeChromatography]

Each antibody was assayed under the following condition.

Mobile phase A: 20 mM MES-NaOH, pH 6.0

Mobile phase B: 20 mM MES-NaOH, 200 mM NaCl, pH 6.0

Column. Bio Pro SP-F (YMC)

Flow rate: 0.5 ml/min

Gradient: 10% B (0-5 min), 10-60% B (5-55 min)

Detection: Abs. 280 nm

FIG. 53 shows a representative chromatogram. The peak at an earlyposition of elution corresponds to B chain-B chain homodimerizedantibody, while the main peak corresponds to A chain-B chainheterodimerized antibody. A chain is introduced with a residuesubstitution (H435R) to reduce the binding to Protein A. The antibodiesused in this experiment are removed in the purification step withrProtein A Sepharose™ Fast Flow (GE Healthcare) during the process ofpreparation by the method described in Reference Example 1. Thus, Achain-A chain homodimerized antibody is hardly detected under thecondition described above. The present inventors calculated:

retention time difference ΔRT(min)=(retention time for heteromericantibody peak)−(retention time for B-chain homomeric antibody peak), asan indicator to assess the separation of heterodimerized andhomodimerized antibodies. Table 72 shows the results of assessment ofvarious variants. The results demonstrate that the retention timedifference between heteromeric antibody and homomeric antibody becomelarger depending on the introduction of designed residue substitutionsand their combinations.[Assessment of the Content of Aggregate by Gel Filtration ChromatographyMethod]

Purified antibodies were assessed for the content of aggregate by SECanalysis using ACQUITY UPLC H-Class system (Waters). 50 mM phosphatebuffer, pH 7.0 containing 300 mM sodium chloride (Isekyu) was used asthe mobile phase and BEH200 SEC (waters) was used as the analyticalcolumn. Measurements were carried out at a wavelength of 215 nm. Thedata was analyzed using Empower2 (Waters). The components eluted on thehigher-molecular-weight side relative to the monomers were collectivelyregarded as the aggregate, and the content was calculated. Table 72shows the assessment results for the various variants. The resultssuggest that the respective variants retain the stability concerningpolymerization, since the content of aggregate was not drasticallyincreased as compared to H240-AK072/H240-BH076/L73-k0 before alteration.

[Assessment of Altered Antibodies for the Midpoint Temperature ofThermal Denaturation (Tm) by Differential Scanning Fluorometry]

Thermal stability of antibodies was assessed by determining theirmidpoint temperature of thermal denaturation (Tm) by differentialscanning fluorometry using Rotor-Gene Q (QIAGEN). This method has beenreported to show an excellent correlation with Tm assessment usingdifferential scanning calorimeter, which is a widely known method forassessing the thermal stability of antibodies (Journal of PharmaceuticalScience 2010, 4:1707-1720).

After 5000 times concentrated SYPRO orange (Molecular Probes) wasdiluted with PBS (Sigma), antibody solutions were mixed with it toprepare measurement samples. 20 μl each of the samples were placed inmeasurement tubes and the temperature was increased from 30° C. up to99° C. The temperature was raised by 0.4° C. and left to stand for about6 seconds, and the fluorescence intensity was determined at 470 nm(excitation wavelength)/555 nm (fluorescent wavelength).

From the data, the temperature at which fluorescence transition wasobserved was calculated as Tm using Rotor-Gene Q Series Software(QIAGEN). In the same manner as reported in Molecular Immunology 37(2000) 697-706 and such, Tm of CH2 domain was defined as Tm1corresponding to the first transition. Meanwhile, Tm values were closebetween CH3 and Fab of the tested antibodies, and it was judgeddifficult to compare them separately. Thus, Tm values used in thisassessment are Tm1 values. Table 72 shows the assessment results forvarious variants. The results suggest that the respective variantsretain the structural stability, because the Tm values were notdrastically reduced as compared to H240-AK072/H240-BH076/L73-k0 beforealteration.

TABLE 72 VARIOUS PHYSICAL PROPERTY DATA Δ RT Tm AGGREGATE Name (min) (°C.) (%) H240-AK072/H240-BH076/L73-k0 5.241 63.0 2.34H240-FA020/H240-FB059/L73-k0 17.283 62.6 1.78H240-FA020/H240-FB082/L73-k0 17.206 62.7 1.50H240-FA020/H240-FB083/L73-k0 19.658 62.5 3.38H240-FA020/H240-FB084/L73-k0 19.433 62.6 3.51H240-FA021/H240-FB059/L73-k0 17.886 62.0 1.28H240-FA021/H240-FB082/L73-k0 17.705 62.2 1.15H240-FA021/H240-FB083/L73-k0 20.203 62.0 2.49H240-FA021/H240-FB084/L73-k0 20.084 62.1 3.05H240-FA030/H240-FB059/L73-k0 24.494 59.9 0.62H240-FA030/H240-FB082/L73-k0 24.624 59.8 0.58H240-FA030/H240-FB083/L73-k0 26.710 59.9 1.96H240-FA030/H240-FB084/L73-k0 26.767 60.0 1.98H240-FA024/H240-FB059/L73-k0 27.505 59.9 0.67H240-FA024/H240-FB082/L73-k0 28.482 59.7 0.63H240-FA024/H240-FB083/L73-k0 31.255 59.7 1.87H240-FA024/H240-FB084/L73-k0 31.171 59.8 1.74[Assessment of Separation in Ion-Exchange Chromatography Purification]

Each sample was assessed for the separation in an ion-exchangechromatography purification method using AKTA avant25 (GE healthcare).The mobile phase used was 20 mM MES buffer, pH 6.0, and 20 mM MESbuffer, pH 6.0, containing 500 mM sodium chloride. The column used wasHi Trap SP HP 1 ml (GE healthcare). Purification was carried out by agradient method using a mixture of two solutions. The purification datawas collected at a wavelength of 280 nm. The result of elution wasassessed using Unicorn6.1 (GE healthcare). FIG. 54 shows an assessmentresult for H240-FA021/H240-BF084/L73-k0 variant. The result demonstratesthat, by introducing the residue substitutions newly identified in thisexperiment, homodimerized and heterodimerized antibodies can beseparated and purified by purifications that use column medium used inlarge scale.

[Example 29] Immunological Assessment of H240-AK072/H240-BH076/L73-k0Variants

[Assessment of the FcγR-Binding Activity by Surface Plasmon ResonanceMethod]

Antibodies of interest were analyzed for the interaction to FcgRaccording to the method described in Reference Example 8.

The assessment results for various variants are shown in Table 73. Theresults demonstrate that the FcγR-binding ability ofH240-FA021/H240-BF084/L73-k0 variant, which was confirmed to beseparated in FIG. 54, was comparable to that ofH240-AK072/H240-BH076/L73-k0 before alteration.

TABLE 73 FcγR BINDING ACTIVITY DATA Name KD (1a) KD (2aR) KD (2aH) KD(2b) KD (3aF) KD (3aV) EXPERIMENT # H240-AK072/H240-BH076/L73-k01.10E−10 2.40E−07 7.20E−08 1.30E−06 2.90E−09 9.90E−10 1H240-FA024/H240-FB059/L73-k0 8.90E−11 2.90E−07 9.30E−08 1.60E−064.40E−09 1.30E−09 1 H240-FA024/H240-FB064/L73-k0 9.50E−11 3.30E−071.10E−07 1.90E−06 4.70E−09 1.30E−09 1 H240-FA024/H240-FB065/L73-k01.50E−10 3.00E−07 9.50E−08 1.70E−06 5.00E−09 1.40E−09 1H240-FA030/H240-FB059/L73-k0 1.20E−10 2.50E−07 8.50E−08 1.50E−064.80E−09 1.40E−09 1 H240-FA030/H240-FB064/L73-k0 5.70E−11 2.90E−079.60E−08 1.80E−06 5.30E−09 1.50E−09 1 H240-FA030/H240-FB065/L73-k01.00E−10 3.40E−07 9.90E−08 2.40E−06 4.60E−09 1.10E−09 1H240-FA012/H240-FB056/L73-k0 1.00E−10 2.80E−07 8.60E−08 1.40E−065.00E−09 1.30E−09 1 H240-FA030/H240-FB059/L73-k0 8.20E−11 2.60E−078.50E−08 1.50E−06 3.90E−09 1.20E−09 1 H240-FA030/H240-FB082/L73-k09.20E−11 3.00E−07 8.80E−08 1.90E−06 4.30E−09 1.30E−09 1H240-FA030/H240-FB083/L73-k0 8.10E−11 2.80E−07 8.20E−08 1.50E−063.90E−09 1.20E−09 1 H240-FA030/H240-FB084/L73-k0 1.20E−10 2.70E−078.80E−08 2.50E−06 4.20E−09 1.30E−09 1 H240-FA024/H240-FB082/L73-k01.30E−10 2.40E−07 8.00E−08 1.50E−06 4.60E−09 1.30E−09 1H240-FA024/H240-FB084/L73-k0 9.50E−11 2.50E−07 7.90E−08 1.50E−064.60E−09 1.20E−09 1 H240-AK072/H240-BH076/L73-k0 1.20E−10 2.80E−076.90E−08 1.50E−06 3.70E−09 1.20E−09 2 H240-FA009/H240-FB001/L73-k01.70E−10 2.50E−07 6.90E−08 1.30E−06 4.30E−09 1.10E−09 2H240-FA012/H240-FB001/L73-k0 7.60E−11 3.50E−07 1.10E−07 1.70E−066.30E−09 1.50E−09 2 H240-FA030/H240-FB001/L73-k0 3.60E−11 4.40E−071.20E−07 3.30E−06 6.40E−09 1.70E−09 2 H240-FA024/H240-FB001/L73-k01.80E−09 3.70E−07 1.10E−07 2.00E−06 6.70E−09 1.90E−09 2H240-FA009/H240-FB017/L73-k0 1.10E−10 2.30E−07 6.10E−08 1.40E−063.00E−09 8.40E−10 2 H240-FA009/H240-FB015/L73-k0 9.30E−11 3.30E−078.40E−08 1.80E−06 4.20E−09 1.30E−09 2 H240-FA009/H240-FB033/L73-k01.20E−10 3.10E−07 8.00E−08 1.60E−06 3.40E−09 1.00E−09 2H240-FA009/H240-FB020/L73-k0 1.20E−10 2.50E−07 6.40E−08 1.40E−063.90E−09 1.10E−09 2 H240-FA009/H240-FB016/L73-k0 1.30E−10 2.50E−077.00E−08 1.40E−06 4.40E−09 1.00E−09 2 H240-FA009/H240-FB052/L73-k05.10E−11 2.70E−07 7.20E−08 1.40E−06 4.30E−09 8.90E−10 2H240-FA009/H240-FB021/L73-k0 5.10E−11 2.80E−07 7.00E−08 2.20E−063.70E−09 7.80E−10 2 H240-FA009/H240-FB047/L73-k0 5.00E−11 2.70E−077.40E−08 1.70E−06 4.40E−09 1.10E−09 2 H240-AK072/H240-BH076/L73-k01.10E−10 2.40E−07 7.90E−08 1.40E−06 3.70E−09 1.20E−09 3H240-FA021/H240-FB059/L73-k0 6.90E−11 2.80E−07 9.20E−08 1.30E−064.40E−09 1.70E−09 3 H240-FA021/H240-FB082/L73-k0 6.00E−11 2.30E−077.70E−08 1.60E−06 4.20E−09 1.50E−09 3 H240-FA021/H240-FB083/L73-k07.80E−11 2.40E−07 6.90E−08 1.30E−06 3.90E−09 1.40E−09 3H240-FA021/H240-FB084/L73-k0 3.30E−11 2.30E−07 6.90E−08 1.30E−063.30E−09 1.30E−09 3 H240-FA020/H240-FB059/L73-k0 3.80E−11 2.50E−077.70E−08 1.30E−06 3.50E−09 1.30E−09 3 H240-FA020/H240-FB082/L73-k07.90E−11 2.70E−07 6.90E−08 1.20E−06 3.40E−09 1.10E−09 3H240-FA020/H240-FB083/L73-k0 1.20E−10 2.30E−07 6.50E−08 1.00E−063.40E−09 1.10E−09 3 H240-FA020/H240-FB084/L73-k0 6.80E−11 2.20E−075.70E−08 1.40E−06 3.50E−09 1.00E−09 3[Immunogenicity Risk Assessment Using in Silico ImmunogenicityPredication Tool, Epibase]

The clinical usefulness and efficacy of antibody pharmaceuticals arelimited by anti-drug antibodies (ADAs). ADAs affect the drug efficacyand kinetics of antibody pharmaceuticals and sometimes cause seriousside effects. Many immunogenicity-influencing factors have beenreported, and in particular it is believed to be important that T cellepitopes are contained in antigens. In silico tools available forpredicting such T cell epitopes include Epibase (Lonza), iTope/TCED(Antitope), and EpiMatrix (EpiVax). It has been reported that sequencescontaining T-cell epitopes present in proteins of interest could bepredicted by using the tools described above (Expert Opin Biol Ther.2007 March; 7(3): 405-18).

Epibase Light (Lonza) is an in silico tool for calculating the bindingcapacity between 9-mer peptide and major DRB1 allele using FASTERalgorism (Expert Opin Biol Ther. 2007 March; 7(3): 405-18). This toolenables identification of T-cell epitopes that strongly or moderatelybind to MHC class II.

An in silico immunogenicity score can be determined for each modifiedantibody according to the following equation (Equation 4) in the systemof Epibase Light (Lonza).Immunogenicity score=Sum(each DRB1 allotype population frequency×numberof critical epitopes)  [Equation 4]

The calculation reflects the abundance ratio of DRB1 allotypes. For thispurpose, it is possible to use the following abundance ratio inCaucasian.

DRB1*1501(24.5%), DRB1*0301(23.7%), DRB1*0701(23.3%), DRB1*0101(15.0%),DRB1*1101(11.6%), DRB1*1302(8.2%), DRB1*1401/1454(4.9%),DRB1*0901(2.3%), DRB1*1502(0.5%), DRB1*1202(0.1%)

All epitopes contained in each modified antibody sequence that exhibitstrong or moderate binding are identified by FASTER algorism, and thenepitopes after excluding human germline sequences and junction sequencesbetween variable region and constant region are used as criticalepitopes in immunogenicity score calculation. When the score is smaller,it means that a sequence has lower immunogenicity risk. Table 74 showsthe risk scores calculated for H240-AK072 and H240-BH076, and variantsobtained therefrom. Thus, variants that result in improved separationand purification capacity for homodimer and heterodimer and whoseimmunogenicity risk is not greatly altered as compared toH240-AK072/H240-BH076/L73-k0 can be produced by selecting anycombination of A chain and B chain.

TABLE 74 IMMUNOGENICITY RISK SCORE Name RISK SCORE H240-AK072 564.6H240-FA020 552.0 H240-FA021 552.0 H240-FA030 565.3 H240-FA024 552.0H240-BH076 480.4 H240-FB059 480.4 H240-FB082 480.4 H240-FB083 505.1H240-FB084 505.1

[Reference Example 1] Construction of Expression Vectors for Antibodiesand Expression and Purification of Antibodies

Amino acid substitutions were introduced by methods known to thoseskilled in the art using QuikChange Site-Directed Mutagenesis Kit(Stratagene), PCR, or In fusion Advantage PCR cloning kit (TAKARA), andexpression vectors were constructed. The nucleotide sequence of theobtained expression vector was determined by methods known to thoseskilled in the art. The produced plasmids were introduced transientlyinto the HEK293H cell line derived from human embryonic kidney cancercells (Invitrogen) or into FreeStyle293 cells (Invitrogen) for antibodyexpression. Antibodies were purified from the obtained culturesupernatant by methods known to those skilled in the art using rProteinA Sepharose™ Fast Flow (GE Healthcare). For the concentration of thepurified antibodies, their absorbance at 280 nm was measured using aspectrophotometer. From the obtained value, the extinction coefficientcalculated by the PACE method was used to calculate the antibodyconcentration (Protein Science 1995; 4: 2411-2423).

[Reference Example 2] Preparation of FcγR and Assessment of BindingActivity to FcγR

Extracellular domains of FcgRs were prepared by the following method.First, a gene of the extracellular domain of FcgR was synthesized by amethod well known to those skilled in the art. At that time, thesequence of each FcgR was produced based on the information registeredat NCBI. Specifically, FcgRI was produced based on the sequence of NCBIAccession # NM_000566.3, FcgRIIa was produced based on the sequence ofNCBI Accession # NM_001136219.1, FcgRIIb was produced based on thesequence of NCBI Accession # NM_004001.3, FcgRIIIa was produced based onthe sequence of NCBI Accession # NM_001127593.1, and FcgRIIIb wasproduced based on the sequence of NCBI Accession # NM_000570.3, and aHis tag was attached to the C terminus. Furthermore, polymorphism isknown for FcgRIIa, FcgRIIIa, and FcgRIIIb, and the polymorphic siteswere produced by referring to J. Exp. Med., 1990, 172: 19-25 forFcgRIIa; J. Clin. Invest., 1997, 100 (5): 1059-1070 for FcgRIIIa; and J.Clin. Invest., 1989, 84, 1688-1691 for FcgRIIIb.

The obtained gene fragments were inserted into an animal cell expressionvector, and expression vectors were produced. The produced expressionvectors were introduced transiently into human embryonic kidney cancercell line-derived FreeStyle293 cells (Invitrogen) to express theproteins of interest. Regarding FcgRIIb used for crystallographicanalysis, the protein of interest was expressed in the presence ofKifunesine at a final concentration of 10 ug/mL, so that the sugar chainadded to FcgRIIb will be the high-mannose type. Cells were cultured, andafter collection of the obtained culture supernatant, this was passedthrough a 0.22 μm filter to obtain the culture supernatant. Inprinciple, the obtained culture supernatants were purified in thefollowing four steps. The steps carried out were, cation exchange columnchromatography (SP Sepharose FF) in step 1, affinity columnchromatography (HisTrap HP) for His tag in step 2, gel filtration columnchromatography (Superdex200) in step 3, and aseptic chromatography instep 4. However, for FcgRI, anion exchange column chromatography using Qsepharose FF was performed as step 1. The purified proteins weresubjected to absorbance measurements at 280 nm using aspectrophotometer; and from the obtained values, the concentrations ofthe purified proteins were calculated using the absorption coefficientcalculated using methods such as PACE (Protein Science 1995; 4:2411-2423).

Analysis of interaction between objective antibody and the FcγR wascarried out using Biacore T100 (GE Healthcare). HBS-EP+ (GE Healthcare)was used as the running buffer, and the measurement temperature was setto 25° C. Chips produced by immobilizing the antigen peptide by theamine coupling method to a Series S Sencor Chip CM5 (GE Healthcare), oralternatively, chips produced by allowing preliminarily biotinylatedantigen peptides to interact with and immobilize onto a Series S SensorChip SA (certified) (GE Healthcare) were used. Antibodies of interestwere captured onto an antigen peptide-immobilized chip, and allowed tointeract to each FcγR diluted with running buffer. To regenerate andrepeatedly use the chip, the antibodies captured on the chip were washedoff by reacting 10 mM glycine-HCl, pH 1.5.

The FcγR-binding activity of each antibody was assessed primarily usingas an indicator the FcγR-binding activity and dissociation constant forFcγR.

The FcγR-binding activity refers to the relative binding activity toFcγR. Regarding the relative FcγR-binding activity, in each measurementthe binding activity of a control sample was taken as 100(%) tocalculate the binding activities of other antibodies. The bindingactivity described above was defined as a value obtained by dividing thelevel of change in the sensorgram before and after interaction of FcγRto the captured antibody, which reflects the binding activity of FcγR,by the quantity of each captured antibody. The reason is that thebinding activity of FcγR depends on the quantity of the capturedantibody.

The dissociation constant of each antibody for FcγR was calculated byperforming kinetic analysis of the result of Biacore measurement.Specifically, to calculate association rate constant ka (L/mol/s) anddissociation rate constant kd (1/s), sensorgrams obtained by measurementwere processed for global fitting by Biacore Evaluation Software using1:1 Langmuir binding model and dissociation constant KD (mol/l) wascalculated from the resulting values.

[Reference Example 3] Production of Expression Vectors forHeterodimerized Antibody Genes and Expression of Each Antibody

As the antibody H chain variable region, those described below wereused:

Q153 (the H chain variable region of anti-human F.IX antibody; SEQ IDNO: 61);

Q407 (the H chain variable region of anti-human F.IX antibody; SEQ IDNO: 62);

J142 (the H chain variable region of anti-human F.X antibody; SEQ ID NO:63);

J300 (the H chain variable region of anti-human F.X antibody; SEQ ID NO:64); and

MRA-VH (the H chain variable region of an anti-human interleukin-6receptor antibody; SEQ ID NO: 65).

As the antibody L chain, those described below were used:

L180-k (the common L chain of anti-human F.IX antibody/anti-human F.Xantibody; SEQ ID NO: 66);

L210-k (the common L chain of anti-human F.IX antibody/anti-human F.Xantibody; SEQ ID NO: 67); and

MRA-k (the L chain of an anti-human interleukin-6 receptor antibody; SEQID NO: 68).

As the antibody H chain constant region, those described below wereused:

G4d (SEQ ID NO: 69), which was constructed from IgG4 by introducing asubstitution mutation from Ser to Pro at position 228 (EU numbering) anddeleting the C-terminal Gly and Lys;

z72 (SEQ ID NO: 70), which was constructed from G4d by introducing asubstitution mutation from His to Arg at position 435 (EU numbering), asubstitution mutation from Tyr to Phe at position 436 (EU numbering),and a substitution mutation from Leu to Pro at position 445 (EUnumbering);z7 (SEQ ID NO: 71), which was constructed from G4d by introducing asubstitution mutation from Glu to Lys at position 356 (EU numbering);z73 (SEQ ID NO: 72), which was constructed from z72 by introducing asubstitution mutation from Lys to Glu at position 439 (EU numbering);z106 (SEQ ID NO: 73), which was constructed from z7 by introducing asubstitution mutation from Lys to Gln at position 196 (EU numbering), asubstitution mutation from Phe to Tyr at position 296 (EU numbering),and a substitution mutation from Arg to Lys at position 409 (EUnumbering);z107 (SEQ ID NO: 74), which was constructed from z73 by introducing asubstitution mutation from Lys to Gln at position 196 (EU numbering), asubstitution mutation from Phe to Tyr at position 296 (EU numbering), asubstitution mutation from Arg to Lys at position 409 (EU numbering),and a substitution mutation from Phe to Tyr at position 436 (EUnumbering); andG1d (SEQ ID NO: 75), which was constructed by deleting the C-terminalGly and Lys from IgG1. The substitution mutation from Glu to Lys atposition 356 (EU numbering) and the substitution mutation from Lys toGlu at position 439 (EU numbering) were introduced for efficientformation of heteromeric molecules from the respective H chains inproducing heteromeric antibodies ((WO 2006/106905) PROCESS FORPRODUCTION OF POLYPEPTIDE BY REGULATION OF ASSEMBLY).

Anti-human F.IX antibody H chain genes Q153-G4d and Q153-z7 wereconstructed by linking respectively G4d and z7 downstream of Q153.Anti-human F.IX antibody H chain gene Q407-z106 was constructed bylinking z106 downstream of Q407. Anti-human F.X antibody H chain geneJ142-G4d, J142-z72, and J142-z73 were constructed by linkingrespectively G4d, z72, and z73 downstream of J142. Anti-human F.Xantibody H chain gene J300-z107 was constructed by linking z107downstream of J300. Anti-human interleukin-6 receptor antibody H chaingene MRA-G1d, MRA-z106, and MRA-z107 were constructed by linkingrespectively G1d, z106, and z107 downstream of MRA-VH.

The respective antibody genes (Q153-G4d, Q153-z7, Q407-z106, J142-G4d,J142-z72, J142-z73, J300-z106, MRA-G1d, MRA-z106, MRA-z107, L180-k,L210-k, and MRA-k) were inserted into animal cell expression vectors.

The following antibodies were transiently expressed in FreeStyle293cells (Invitrogen) by transfection using the constructed expressionvectors. As seen below, multiple antibody genes to be transfected werearranged and used as antibody names.

MRA-G1d/MRA-k

MRA-z106/MRA-z107/MRA-k

Q153-G4d/J142-G4d/L180-k

Q153-G4d/J142-z72/L180-k

Q153-z7/J142-z73/L180-k

Q407-z106/J300-z107/L210-k

[Reference Example 4] Assessment of Elution Condition forHeterodimerized Antibody in Protein A Affinity Chromatography, andSeparation and Purification

A FreeStyle293 cell culture medium (hereinafter abbreviated as CM)obtained by transient expression of Q153-G4d/J142-G4d/L180-k andQ153-G4d/J142-z72/L180-k was used as a sample to assess the elutioncondition in Protein A affinity chromatography. CM filtered throughφ0.22-μm filter was loaded onto rProtein A Sepharose Fast Flow column(GE Healthcare) equilibrated with D-PBS. Washes 1 and 2, and Elutions 1to 5 shown in Table 75 were performed in a stepwise manner. The quantityof CM to be loaded was adjusted in a way that the quantity of antibodyloaded onto the column is 20 mg/ml resine. Fractions eluted under eachcondition were collected and the components in each elution fractionwere identified by cation exchange chromatography analysis. To preparecontrols, each CM was loaded onto rProtein G Sepharose Fast Flow resin(GE Healthcare). Samples purified by batchwise elution were used ascontrols. Since Protein G binds to the Fab domain of an antibody, allantibody species (bispecific antibody of interest in which two types ofH chains are associated in a heteromeric manner (heteromeric antibody)and as an impurity monospecific homomeric antibodies in whichsingle-type H chains are homomerically associated) in CM can be purifiedby using protein G, regardless of their Protein A-binding activity.

TABLE 75 EQUILIBRATION D-PBS WASH 1 400 mM Arg-HCl/D-PBS WASH 2 20 mMNaCitrate, pH 5.0 ELUTION 1 20 mM NaCitrate, pH 4.0 ELUTION 2 20 mMNaCitrate, pH 3.8 ELUTION 3 20 mM NaCitrate, pH 3.6 ELUTION 4 20 mMNaCitrate, pH 3.4 ELUTION 5 20 mM NaCitrate, pH 3.2

CM in which Q153-G4d/J142-G4d/L180-k or Q153-G4d/J142-z72/L180-k hadbeen expressed was eluted from a protein A column (Elutions 1 to 5), andthe respective eluted fractions were analyzed by cation exchangechromatography. As for Q153-G4d/J142-G4d/L180-k, the analysis revealedthat as the elution condition was altered from 1 to 5, i.e., as the pHof the elution buffer was reduced, the antibody composition of theeluted fractions changed gradually in the order from the homomericantibody J142-G4d/L180-k to the heteromeric antibodyQ153-G4d/J142-G4d/L180-k, and then to the homomeric antibodyQ153-G4d/L180-k. The order of elution is understood to be in accordancewith the strength of binding to Protein A. This implies that the ProteinA-binding strength of homomeric antibody Q153-G4d/L180-k, which remainedbound until a lower pH, is greater than homomeric speciesJ142-G4d/L180-k (homomeric antibody against FX) which eluted at a highpH. Variable region J142 is known to be a sequence that does not bind toProtein A. Specifically, homomeric species J142-G4d/L180-k (homomericantibody against FX) has two Protein A-binging sites; heteromericantibody Q153-G4d/J142-G4d/L180-k has three Protein A-binging sites; andhomomeric antibody Q153-G4d/L180-k (homomeric antibody against FIX) hasfour Protein A-binging sites. It was thus demonstrated that, moreProtein A-binding sites resulted in stronger Protein A binding and thusa lower pH was required for elution.

Meanwhile, as for Q153-G4d/J142-z72/L180-k, it was revealed that as theelution condition was altered from 1 to 5, the antibody composition inthe eluted fraction changed from the heteromeric antibodyQ153-G4d/J142-z72/L180-k to the homomeric antibody Q153-G4d/L180-k.Homomeric antibody J142-z72/L180-k (homomeric antibody against FX) wasalmost undetectable in each elution fraction, and this indicates that ithas no Protein A-binding ability. It is thought that the lack of proteinA-binding ability of J142-z72 might be due to the introducedsubstitution mutation of Arg for His at position 435 (EU numbering).Homomeric antibody J142-z72/L180-k (homomeric antibody against FX) hasno Protein A-binding site; heteromeric antibody Q153-G4d/J142-z72/L180-khas two Protein A-binding sites; and homomeric antibody Q153-G4d/L180-k(homomeric antibody against FIX) has four Protein A-binding sites. Sincehomomeric antibody J142-z72/L180-k (homomeric antibody against FX)flowed through without binding to Protein A and was not detected in eachelution fraction. Furthermore, in both cases of Q153-G4d/J142-G4d/L180-kand Q153-G4d/J142-z72/L180-k, it was suggested that the heteromericantibody and homomeric antibody Q153-G4d/L180-k (a homomeric antibodyagainst FIX) were separable from each other at pH 3.6 or a lower pH.

Heterodimerized antibodies were purified by Protein A columnchromatography under the purification conditions assessed as describedabove.

CM of the antibodies listed below were used as a sample.

Q153-G4d/J142-G4d/L180-k

Q153-G4d/J142-z72/L180-k

Q153-z7/J142-z73/L180-k

Q407-z106/J300-z107/L210-k

CM filtered through φ0.22-μm filter was loaded onto rProtein A SepharoseFast Flow column (GE Healthcare) equilibrated with D-PBS. Washes 1 and 2and Elutions 1 and 2 shown in Table 76 were carried out (Elution 1 alonewas performed for Q407-z106/J300-z107/L210-k). The elution condition wasbased on the result described above. The quantity of CM to be loaded wasadjusted in a way that the quantity of the loaded antibody is 20 mg/mlresine. Fractions eluted under each condition were collected and thecomponents contained in each elution fraction were identified by cationexchange chromatography analysis. In the same manner as shown in theresult described above, each CM was loaded onto rProtein G SepharoseFast Flow resin (GE Healthcare) to prepare controls. Samples purified bybatchwise elution were used as controls.

TABLE 76 EQUILIBRATION D-PBS WASH 1 400 mM Arg-HCl/D-PBS WASH 2 20 mMNaCitrate, pH 5.0 ELUTION 1 20 mM NaCitrate, pH 3.6 ELUTION 2 20 mMNaCitrate, pH 2.7

The results of cation exchange chromatography analysis for each elutedfraction are shown in Tables 77 to 80 below. The values represent thearea of elution peak expressed in percentage. With respect to antibodiesother than Q153-G4d/J142-G4d/L180-k, the homomeric antibody against FXwas almost undetectable in any elution fraction. It was revealed thatnot only homomeric antibody J142-z72 (homomeric antibody against FX) butalso homomeric antibodies J142-z73 and J300-z107 (homomeric antibodiesagainst FX) did not bind to Protein A. It is thought that the lack ofprotein A-binding ability in the homomeric antibody against FX was dueto the substitution mutation of Arg for His at position 435 (EUnumbering), which was introduced into the H chain constant region of theantibody against FX. The heteromeric antibody, which is a bispecificantibody of interest, was mostly detected in the fraction of elution 1,while, although they were also detected at a very low level in thefraction of Elution 1, the homomeric antibody against FIX was mostlyeluted by Elution 2. As compared to Q153-G4d/J142-z72/L180-k, in thecases of Q153-z7/J142-z73/L180-k and Q407-z106/J300-z107/L210-k, theproportion of the heteromeric antibody (bispecific antibody of interest)was considerably increased in the fraction eluted at pH 3.6. It wasdemonstrated that when a substitution mutation from Glu to Lys atposition 356 (EU numbering) and a substitution mutation from Lys to Gluat position 439 (EU numbering) for the purpose of efficient formation ofheteromeric molecules for respective H chains were introduced inaddition to the substitution mutation from His to Arg at position 435(EU numbering), the heteromeric antibody, which is a bispecific antibodyof interest, could be purified to a purity of 98% or higher through theProtein A-based purification step alone.

The finding described above shows that, based on the difference in thenumber of Protein A-binding sites between homomeric antibody andheteromeric antibody, the heteromeric antibody can be efficientlyseparated and purified to high purity using Protein A chromatographystep alone.

TABLE 77 Q153-G4d/J142-G4d/L180-k pH 3.6 pH 2.7 ELUTION ELUTION PEAKAREA (%) CONTROL FRACTION FRACTION J142-G4d/L180-k 17.6 27.5 —Q153-G4d/J142-G4d/L180-k 48.3 58.4 9.0 Q153-G4d/L180-k 34.1 14.1 91.0

TABLE 78 Q153-G4d/J142-z72/L180-k pH 3.6 pH 2.7 ELUTION ELUTION PEAKAREA (%) CONTROL FRACTION FRACTION J142-z72/L180-k 8.4 0.9 —Q153-G4d/J142-z72/L180-k 50.8 81.0 2.2 Q153-G4d/L180-k 40.8 18.1 97.8

TABLE 79 Q153-z7/J142-z73/L180-k pH 3.6 pH 2.7 ELUTION ELUTION PEAK AREA(%) CONTROL FRACTION FRACTION J142-z73/L180-k 3.2 — —Q153-z7/J142-z73/L180-k 90.7 98.1 2.7 Q153-z7/L180-k 6.1 1.9 97.3

TABLE 80 Q407-z106/J300-z107/L210-k pH 3.6 pH 2.7 ELUTION ELUTION PEAKAREA (%) CONTROL FRACTION FRACTION J300-z107/L210-k 5.8 —Q407-z106/J300-z107/L210-k 84.6 98.9 Q407-z106/L210-k 9.7 1.1

[Reference Example 5] Assessment of Altered Antibodies for Tm byDifferential Scanning Fluorometry

In this assessment, Tm of altered antibodies was evaluated bydifferential scanning fluorometry using Rotor-Gene Q (QIAGEN). Thismethod has been reported to show an excellent correlation with Tmassessment using differential scanning calorimeter, which is a widelyknown method for assessing the thermal stability of antibodies (Journalof Pharmaceutical Science 2010, 4: 1707-1720).

After 5000 times concentrated SYPRO orange (Molecular Probes) wasdiluted with PBS (Sigma), antibody solutions were mixed with it toprepare measurement samples. 20 μL each of the samples were placed inmeasurement tubes and the temperature was increased at a temperatureraising rate of 240° C./hr from 30° C. up to 99° C. The fluorescencechange with increasing temperature was detected at 470 nm (excitationwavelength)/555 nm (fluorescent wavelength).

From the data, the temperature at which fluorescence transition wasobserved was calculated as Tm using Rotor-Gene Q Series Software(QIAGEN).

[Reference Example 6] Acceleration Test of Modified HeteromericAntibodies

Acceleration tests were performed regarding the antibodies described inthis Example and the storage stability was compared.

After purification with Protein A, antibodies were prepared at 1.0 mg/mlusing PBS containing 0.2 mM hydrochloric acid, and stored in anincubator at 40° C. At the start of storage, after two weeks of storage,and after four weeks of storage, each antibody was assessed for themonomer content by size exclusion chromatography using G3000 SW_(XL)column.

[Reference Example 7] ADCC Activity of Each Test Antibody Using HumanPeripheral Mononuclear Cells as Effector Cells

Variants with FcγR-binding activity increased by introducing analteration into only one H chain of an antibody were assayed for ADCCactivity according to the method described below.

ADCC of each test antibody was assayed using human peripheral bloodmononuclear cells (hereinafter referred to as “human PBMC”) as effectorcells by the procedure described below.

(1) Preparation of Human PBMC Solutions

Using syringes pre-filled with 200 μl of 1,000 units/ml heparin solution(Novo-Heparin 5000 units for Injection; Novo Nordisk), 50 ml ofperipheral blood was collected from healthy volunteers (male adult)affiliated with Chugai Pharmaceutical Co. Ltd. The peripheral blood wasdiluted two-fold with PBS(−), and divided into four equal parts, each ofwhich was transferred into a pre-centrifuged leukocyte separation tubeLeucosep (Greiner Bio-One) containing 15 ml of Ficoll-Paque PLUS. Theseparation tubes containing an aliquot of the peripheral blood werecentrifuged at 2,150 rpm and room temperature for ten minutes. Then, theresulting mononuclear cell fractions were collected. The cells in eachfraction was washed once with Dulbecco's Modified Eagle's Medium (SIGMA)containing 10% FBS (hereinafter referred to as “10% FBS/D-MEM”), andthen suspended at a density of 5×10⁶ cells/ml in 10% FBS/D-MEM. The cellsuspensions were used as human PBMC solutions in the subsequentexperiments.

(2) Preparation of Target Cells

SK-pca13a resulting from forced expression of human glypican-3 inSK-Hep-1 was detached from the dish, and 1.85 MBq of Cr-51 was added to3×10⁶ cells. Cells added with Cr-51 were incubated in an incubator under5% carbon dioxide gas at 37° C. for one hour. Then the cells were washedonce with 10% FBS/D-MEM, and suspended at a cell density of 2×10⁵cells/ml in 10% FBS/D-MEM. The cell suspension was used as target cellsin the subsequent experiments.

(3) Chromium Release Test (ADCC Activity)

ADCC activity was assessed based on specific chromium release ratedetermined by chromium release assay. First, antibody solutions preparedat each concentration (0, 0.004, 0.04, 0.4, 4, and 40 μg/ml) were addedin 50 μl to respective wells of a 96-well U-bottomed plate. Then, 50μl-aliquots of the target cells prepared as described in (2) were plated(1×10⁴ cells/well) and allowed to stand at room temperature for 15minutes. 100 μl of human PBMC solution prepared as described in (1) wasadded to each well (5×10⁵ cells/well). The plates were incubated under5% carbon dioxide gas in a CO₂ incubator at 37° C. for four hours, andthen centrifuged. 100 μl of culture supernatant in each well of theplates was measured for radioactivity using a gamma counter. Thespecific chrome release rate was determined by the following formula:[Specific chrome release rate (%)]=(A−C)×100/(B−C).

In this formula, “A” represents mean radioactivity (cpm) of 100 μl ofculture supernatant in each well. “B” represents mean radioactivity(cpm) of 100 μl of culture supernatant in a well containing targetcells, 100 μl of 2% NP-40 aqueous solution (Nonidet P-40; NacalaiTesques), and 50 μl of 10% FBS/D-MEM. Furthermore, “C” represents meanradioactivity (cpm) of 100 μl of culture supernatant in a wellcontaining target cells and 150 μl of 10% FBS/D-MEM. The test wasconducted in triplicate. The mean and standard deviation of the specificchrome release rate (%) which reflects the ADCC of each test antibodywere calculated based on the assay described above.

[Reference Example 8] Preparation of FcγR and Assessment of BindingActivity to FcγR

Extracellular domains of FcgRs were prepared by the following method.First, a gene of the extracellular domain of FcgR was synthesized by amethod well known to those skilled in the art. At that time, thesequence of each FcgR was produced based on the information registeredat NCBI. Specifically, FcgRI was produced based on the sequence of NCBIAccession # NM_000566.3, FcgRIIa was produced based on the sequence ofNCBI Accession # NM_001136219.1, FcgRIIb was produced based on thesequence of NCBI Accession # NM_004001.3, FcgRIIIa was produced based onthe sequence of NCBI Accession # NM_001127593.1, and FcgRIIIb wasproduced based on the sequence of NCBI Accession # NM_000570.3, and aHis tag was attached to the C terminus. Furthermore, polymorphism isknown for FcgRIIa, FcgRIIIa, and FcgRIIIb, and the polymorphic siteswere produced by referring to J. Exp. Med., 1990, 172: 19-25 forFcgRIIa; J. Clin. Invest., 1997, 100 (5): 1059-1070 for FcgRIIIa; and J.Clin. Invest., 1989, 84, 1688-1691 for FcgRIIIb.

The obtained gene fragments were inserted into an animal cell expressionvector, and expression vectors were produced. The produced expressionvectors were introduced transiently into human embryonic kidney cancercell line-derived FreeStyle293 cells (Invitrogen) to express theproteins of interest. Regarding FcgRIIb used for crystallographicanalysis, the protein of interest was expressed in the presence ofKifunesine at a final concentration of 10 ug/mL, so that the sugar chainadded to FcgRIIb will be the high-mannose type. Cells were cultured, andafter collection of the obtained culture supernatant, this was passedthrough a 0.22 μm filter to obtain the culture supernatant. Inprinciple, the obtained culture supernatants were purified in thefollowing four steps. The steps carried out were, cation exchange columnchromatography (SP Sepharose FF) in step 1, affinity columnchromatography (HisTrap HP) for His tag in step 2, gel filtration columnchromatography (Superdex200) in step 3, and aseptic chromatography instep 4. However, for FcgRI, anion exchange column chromatography using Qsepharose FF was performed as step 1. The purified proteins weresubjected to absorbance measurements at 280 nm using aspectrophotometer; and from the obtained values, the concentrations ofthe purified proteins were calculated using the absorption coefficientcalculated using methods such as PACE (Protein Science 1995; 4:2411-2423).

Analysis of interaction between objective antibody and the FcγR wascarried out using Biacore T100 (GE Healthcare), Biacore T200, BiacoreA100, and Biacore 4000. HBS-EP+ (GE Healthcare) was used as the runningbuffer, and the measurement temperature was set to 25° C. Chips producedby immobilizing the antigen peptide by the amine coupling method to aSeries S sensor Chip CM5 (GE Healthcare), chips produced by allowingpreliminarily biotinylated antigen peptides to interact with andimmobilize onto a Series S Sensor Chip SA (certified) (GE Healthcare),chips produced by immobilizing Protein L (ACTIGEN, BioVision) to SeriesS Sencor Chip CM5 (GE Healthcare), or chips produced by immobilizingProtein A/G (Thermo Scientific) to Series S Sencor Chip CM5 (GEHealthcare) were used. Antibodies of interest were captured onto thesechips, and allowed to interact to each FcγR diluted with running buffer.To regenerate and repeatedly use the chip, the antibodies captured ontothe chip were washed off by reacting 10 mM glycine-HCl, pH 1.5.

The FcγR-binding activity of each antibody was assessed primarily usingas an indicator the FcγR-binding activity and dissociation constant forFcγR.

The FcγR-binding activity refers to the relative binding activity toFcγR. Regarding the relative FcγR-binding activity, in each measurementthe binding activity of a control sample was taken as 100(%) tocalculate the binding activities of other antibodies. The bindingactivity described above was defined as a value obtained by dividing thelevel of change in the sensorgram before and after interaction of FcγRto the captured antibody, which reflects the binding activity of FcγR,by the quantity of each captured antibody. The reason is that thebinding activity of FcγR depends on the quantity of the capturedantibody.

The dissociation constant of each antibody for FcγR was calculated byperforming kinetic analysis of the result of Biacore measurement.Specifically, to calculate association rate constant ka (L/mol/s) anddissociation rate constant kd(1/s), sensorgrams obtained by measurementwere processed for global fitting by Biacore Evaluation Software using1:1 Langmuir binding model. Dissociation constant KD (mol/l) wascalculated from the resulting values.

[Reference Example 9] ADCC Activity of Each Test Antibody Using HumanPeripheral Blood Mononuclear Cells as Effector Cells

Each variant with Fc alteration is assayed for their ADCC activityaccording to the method described below.

ADCC activity of each test antibody was assayed using human peripheralblood mononuclear cells (hereinafter referred to as “human PBMC”) aseffector cells by the procedure described below.

(1) Preparation of Human PBMC Solutions

Using syringes pre-filled with 200 μl of 1,000 units/ml heparin solution(Novo-Heparin 5000 units for Injection; Novo Nordisk), 50 ml ofperipheral blood was collected from healthy volunteers (male adult)affiliated with Chugai Pharmaceutical Co. Ltd. The peripheral blood wasdiluted two-fold with PBS(−), and divided into four equal parts, each ofwhich was transferred into a pre-centrifuged leukocyte separation tubeLeucosep (Greiner Bio-One) containing 15 ml of Ficoll-Paque PLUS. Theseparation tubes containing an aliquot of the peripheral blood werecentrifuged at 2,150 rpm and room temperature for ten minutes. Then, theresulting mononuclear cell fractions were collected. The cells in eachfraction was washed once with Dulbecco's Modified Eagle's Medium (SIGMA)containing 10% FBS (hereinafter referred to as “10% FBS/D-MEM”), andthen suspended at a density of 5×10⁶ cells/ml or 2.5×10⁶ cells/ml in 10%FBS/D-MEM. The cell suspensions were used as human PBMC solutions in thesubsequent experiments.

(2) Preparation of Target Cells

SK-pca13a resulting from forced expression of human Epiregulin inSK-Hep-1, SKE18, human colon cancer cell line DLD-1, or human pancreascell line MIAPaCa-2 were detached from the dish, and 200 μL of 0.2 mg/mLCalcein solution was added to 1×10⁶ cells or 1.85 MBq of Cr-51 was addedto 3×10⁶ cells. Cells added with Calcein or Cr-51 were incubated in anincubator under 5% carbon dioxide gas at 37° C. for one or two hours.Then, the cells were washed once with 10% FBS/D-MEM, and suspended at acell density of 2×10⁵ cells/ml in 10% FBS/D-MEM. The cell suspension wasused as target cells in the subsequent experiments.

(3-1) Calcein or Chromium Release Assay (ADCC Activity)

ADCC activity was assessed based on specific Calcein or chromium releaserate determined by Calcein or chromium release assay. First, antibodysolutions prepared at each concentration (0, 0.004, 0.04, 0.4, 4, and 40μg/ml) were added in 50 μl to respective wells of a 96-well U-bottomedplate. Then, 50 μl-aliquots of the target cells prepared as described in(2) were plated (1×10⁴ cells/well) and allowed to stand at roomtemperature for 15 minutes. 100 μl of human PBMC solution prepared asdescribed in (1) was added to each well (5×10⁵ cells/well or 2.5×10⁵cells/well). The plates were incubated under 5% carbon dioxide gas in aCO₂ incubator at 37° C. for four hours, and then centrifuged. 100 μl ofculture supernatant in each well of the plates was measured for Calceinfluorescence and radioactivity using a absorption meter and gammacounter. The specific chrome release rate was determined by thefollowing formula:[Specific Calcein or chrome release rate (%)]=(A−C)×100/(B−C).

In this formula, “A” represents mean Calcein fluorescence (excitationwavelength: 485 nm; fluorescence wavelength: 535 nm) or meanradioactivity (cpm) of 100 μl of culture supernatant in each well. “B”represents mean Calcein fluorescence (excitation wavelength: 485 nm;fluorescence wavelength: 535 nm) or mean radioactivity (cpm) of 100 μlof culture supernatant in a well containing target cells, 100 μl of 2%NP-40 aqueous solution (Nonidet P-40; Nacalai Tesques), and 50 μl of 10%FBS/D-MEM. Furthermore, “C” represents mean Calcein fluorescence(excitation wavelength: 485 nm; fluorescence wavelength: 535 nm) or meanradioactivity (cpm) of 100 μl of culture supernatant in a wellcontaining target cells and 150 μl of 10% FBS/D-MEM. The test wasconducted in triplicate. The mean and standard deviation of the specificCalcein or chrome release rate (%) which reflects the ADCC of each testantibody were calculated based on the assay described above.

The invention claimed is:
 1. A molecule comprising a heterodimeric Fcregion that is a heterodimer of a first polypeptide comprising a firstCH2 domain and a second polypeptide comprising a second CH2 domain thatdiffers in amino acid sequence from the first CH2 domain, wherein thefirst CH2 domain is an IgG1 CH2 domain comprising a set of amino acidmutations selected from sets (i)-(vi) (all positions by EU numbering):(i) substitution of L at position 234 with Y, substitution of L atposition 235 with Y, substitution of G at position 236 with W,substitution of H at position 268 with D, and substitution of S atposition 298 with A; (ii) substitution of L at position 234 with Y,substitution of L at position 235 with Y, substitution of G at position236 with W, substitution of H at position 268 with D, substitution of Dat position 270 with E, and substitution of S at position 298 with A;(iii) substitution of L at position 234 with Y, substitution of L atposition 235 with Q, substitution of G at position 236 with W,substitution of S at position 239 with M, substitution of H at position268 with D, substitution of D at position 270 with E, and substitutionof S at position 298 with A; (iv) substitution of L at position 234 withY, substitution of L at position 235 with Y, substitution of G atposition 236 with W, substitution of H at position 268 with D,substitution of S at position 298 with A, and substitution of A atposition 327 with D; (v) substitution of L at position 234 with Y,substitution of L at position 235 with Y, substitution of G at position236 with W, substitution of S at position 239 with M, substitution of Hat position 268 with D, substitution of S at position 298 with A, andsubstitution of A at position 327 with D; (vi) substitution of L atposition 234 with Y, substitution of L at position 235 with Y,substitution of G at position 236 with W, substitution of S at position239 with M, substitution of H at position 268 with D, substitution of Sat position 298 with A, substitution of A at position 327 with D,substitution of L at position 328 with W, and substitution of K atposition 334 with L; and the second CH2 domain is an IgG1 CH2 domaincomprising a set of amino acid mutations selected from sets (vii)-(ix)(all positions by EU numbering): (vii) substitution of K at position 326with D, substitution of A at position 330 with M, and substitution of Kat position 334 with E; (viii) substitution of D at position 270 with E,substitution of K at position 326 with D, substitution of A at position330 with M, and substitution of K at position 334 with E; (ix)substitution of D at position 270 with E, substitution of K at position326 with D, substitution of A at position 330 with K, and substitutionof K at position 334 with E.
 2. The molecule of claim 1, wherein theamino acid sequences of the first and the second polypeptides differ atone or more other positions in addition to the positions of the set ofmutations in the first CH2 domain and the positions of the set ofmutations in the second CH2 domain, wherein the differences at the oneor more other positions confer or increase a difference in isoelectricpoints between the first polypeptide and the second polypeptide.
 3. Themolecule of claim 2, wherein each of the first and second polypeptidescomprises a human IgG1 Fc region with multiple mutations, and whereinthe one or more other positions where the first and second polypeptidesdiffer in sequence are selected from the following positions in one orboth of the human IgG1 Fc regions (all positions by EU numbering): 137,138, 139, 147, 192, 193, 196, 198, 199, 203, 214, 263, 272, 274, 278,288, 290, 316, 317, 320, 324, 335, 337, 340, 358, 360, 362, 364, 383,384, 385, 386, 387, 390, 397,
 422. 4. The molecule of claim 2, whereineach of the first and second polypeptides comprises a human IgG1 Fcregion with multiple mutations, and wherein the one or more otherpositions where the first and second polypeptides differ in sequence areselected from: one or more of the following positions in the human IgG1Fc region of the first polypeptide (all positions by EU numbering): 196,199, 263, 272, 316, 358, 364, 383, 387, 397; and one or more of thefollowing positions in the human IgG1 Fc region of the secondpolypeptide (all positions by EU numbering): 137, 138, 139, 147, 192,193, 198, 199, 203, 214, 274, 278, 288, 290, 316, 317, 320, 324, 335,337, 340, 358, 360, 362, 383, 384, 385, 386, 390,
 422. 5. The moleculeof claim 1, wherein the molecule is an antibody.
 6. The molecule ofclaim 5, wherein the antibody is a bispecific antibody.
 7. The moleculeof claim 1, wherein the molecule is a peptide Fc fusion protein or ascaffold Fc fusion protein.
 8. A pharmaceutical composition comprisingthe molecule of claim 1 and a medically acceptable carrier.
 9. A methodof producing the molecule of claim 1, the method comprising: producingthe first polypeptide and the second polypeptide; and generating themolecule comprising a heterodimeric Fc region that is a heterodimer ofthe first polypeptide and the second polypeptide.
 10. The method ofclaim 9, further comprising: assaying a function of the generatedmolecule, thereby determining that said function of the generatedmolecule is increased compared to said function of both (a) a dimeric Fcregion that is a homodimer of the first polypeptide, and (b) a dimericFc region that is a homodimer of the second polypeptide, wherein thefunction is (i) binding affinity for an Fc receptor, or (ii) selectivityof binding to one type of Fc receptor over another type of Fc receptor.11. A method of producing the molecule of claim 1, the methodcomprising: identifying a parent molecule comprising an Fc region thatis a homodimer of a starting polypeptide; producing a first mutatedversion of the starting polypeptide, the first mutated version being thefirst polypeptide; producing a second mutated version of the startingpolypeptide, the second mutated version being the second polypeptide;and generating the molecule comprising a heterodimeric Fc region that isa heterodimer of the first polypeptide and the second polypeptide. 12.The method of claim 11, further comprising: assaying a function of thegenerated molecule, thereby determining that said function of thegenerated molecule is increased (a) compared to said function of adimeric Fc region that is a homodimer of the first polypeptide, and (b)compared to said function of a dimeric Fc region that is a homodimer ofthe second polypeptide, wherein the function is (i) binding affinity foran Fc receptor, or (ii) selectivity of binding to one type of Fcreceptor over another type of Fc receptor.
 13. The molecule of claim 1,wherein the first CH2 domain comprises the mutations of set (i).
 14. Themolecule of claim 1, wherein the first CH2 domain comprises themutations of set (ii).
 15. The molecule of claim 1, wherein the firstCH2 domain comprises the mutations of set (iii).
 16. The molecule ofclaim 1, wherein the first CH2 domain comprises the mutations of set(iv).
 17. The molecule of claim 1, wherein the first CH2 domaincomprises the mutations of set (v).
 18. The molecule of claim 1, whereinthe first CH2 domain comprises the mutations of set (vi).
 19. Themolecule of claim 1, wherein the second CH2 domain comprises themutations of set (vii).
 20. The molecule of claim 1, wherein the secondCH2 domain comprises the mutations of set (viii).
 21. The molecule ofclaim 1, wherein the second CH2 domain comprises the mutations of set(ix).
 22. The molecule of claim 1, wherein the first CH2 domaincomprises the mutations of set (i) and the second CH2 domain comprisesthe mutations of set (vii).
 23. The molecule of claim 1, wherein thefirst CH2 domain comprises the mutations of set (i) and the second CH2domain comprises the mutations of set (viii).
 24. The molecule of claim1, wherein the first CH2 domain comprises the mutations of set (i) andthe second CH2 domain comprises the mutations of set (ix).
 25. Themolecule of claim 1, wherein the first CH2 domain comprises themutations of set (ii) and the second CH2 domain comprises the mutationsof set (vii).
 26. The molecule of claim 1, wherein the first CH2 domaincomprises the mutations of set (ii) and the second CH2 domain comprisesthe mutations of set (viii).
 27. The molecule of claim 1, wherein thefirst CH2 domain comprises the mutations of set (ii) and the second CH2domain comprises the mutations of set (ix).
 28. The molecule of claim 1,wherein the first CH2 domain comprises the mutations of set (iii) andthe second CH2 domain comprises the mutations of set (vii).
 29. Themolecule of claim 1, wherein the first CH2 domain comprises themutations of set (iii) and the second CH2 domain comprises the mutationsof set (viii).
 30. The molecule of claim 1, wherein the first CH2 domaincomprises the mutations of set (iii) and the second CH2 domain comprisesthe mutations of set (ix).
 31. The molecule of claim 1, wherein thefirst CH2 domain comprises the mutations of set (iv) and the second CH2domain comprises the mutations of set (vii).
 32. The molecule of claim1, wherein the first CH2 domain comprises the mutations of set (iv) andthe second CH2 domain comprises the mutations of set (viii).
 33. Themolecule of claim 1, wherein the first CH2 domain comprises themutations of set (iv) and the second CH2 domain comprises the mutationsof set (ix).
 34. The molecule of claim 1, wherein the first CH2 domaincomprises the mutations of set (v) and the second CH2 domain comprisesthe mutations of set (vii).
 35. The molecule of claim 1, wherein thefirst CH2 domain comprises the mutations of set (v) and the second CH2domain comprises the mutations of set (viii).
 36. The molecule of claim1, wherein the first CH2 domain comprises the mutations of set (v) andthe second CH2 domain comprises the mutations of set (ix).
 37. Themolecule of claim 1, wherein the first CH2 domain comprises themutations of set (vi) and the second CH2 domain comprises the mutationsof set (vii).
 38. The molecule of claim 1, wherein the first CH2 domaincomprises the mutations of set (vi) and the second CH2 domain comprisesthe mutations of set (viii).
 39. The molecule of claim 1, wherein thefirst CH2 domain comprises the mutations of set (vi) and the second CH2domain comprises the mutations of set (ix).